Process and apparatus for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars

ABSTRACT

The invention relates to a process for the preparation of a sugar product from lignocellulosic material, comprising the following steps:
         a) optionally, pretreatment of the lignocellulosic material;   b) optionally, washing of the optionally pretreated lignocellulosic material;   c) enzymatic hydrolysis of the optionally washed and/or optionally pretreated lignocellulosic material in a hydrolysis reactor using an enzyme composition comprising at least two cellulase; and   d) optionally, recovery of a sugar product;
 
wherein during the enzymatic hydrolysis oxygen-containing gas is added to the lignocellulosic material in the hydrolysis reactor and wherein part of the oxygen-containing gas, added to the lignocellulosic material, is gas originating from the headspace of the reactor,
 
preferably during part of the time of the enzymatic hydrolysis less oxygen is added to the lignocellulosic material compared to the other part of the time of the enzymatic hydrolysis, or preferably during part of the time of the enzymatic hydrolysis no oxygen is added to the lignocellulosic material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/300,278, filed 28 Sep. 2016, which is a National Stage entry ofInternational Application No. PCT/EP2015/051839, filed Jan. 29, 2015,which claims priority to European Patent Application No. 14163359.4,filed Apr. 3, 2014, European Patent Application No. 14166539.8, filedApr. 30, 2014, and European Patent Application No. 14166545.5, filedApr. 30, 2014. The disclosures of the priority applications areincorporated in their entirety herein by reference.

FIELD OF THE INVENTION

The invention relates to an apparatus and a process for the enzymatichydrolysis of lignocellulosic material and fermentation of sugars.

BACKGROUND OF THE INVENTION

Lignocellulosic plant material, herein also called feedstock, is arenewable source of energy in the form of sugars that can be convertedinto valuable products e.g. sugars or bio-fuel, such as bioethanol.During this process, (ligno- or hemi-)cellulose present in thefeedstock, such as wheat straw, corn stover, rice hulls, etc., isconverted into reducing sugars by (hemi)cellulolytic enzymes, which thenare optionally converted into valuable products such as ethanol bymicroorganisms like yeast, bacteria and fungi.

Since the (hemi)cellulose is crystalline and entrapped in a network oflignin, the conversion into reducing sugars is in general slow andincomplete. Typically, enzymatic hydrolysis of untreated feedstockyields sugars <20% of theoretical quantity. By applying a chemical andthermo-physical pretreatment, the (hemi)cellulose is more accessible forthe (hemi)cellulolytic enzymes, and thus conversions go faster and athigher yields.

A typical ethanol yield from glucose, derived from pretreated cornstover, is 40 gallons of ethanol per 1000 kg of dry corn stover (Badger,P, Ethanol from cellulose: a general review, Trends in new crops and newuses, 2002, J. Janick and A. Whipkey (eds.) ASHS Press, Alexandria, Va.)or 0.3 g ethanol per g feedstock. The maximum yield of ethanol oncellulose base is approximately 90%.

Cellulolytic enzymes—most of them are produced by species likeTrichoderma, Humicola and Aspergillus—are commercially used to convertpretreated feedstock into a mash containing insoluble (hemi)cellulose,reducing sugars made thereof, and lignin. Thermostable cellulolyticenzymes derived from Rasamsonia, have been used for degradinglignocellulosic feedstock and these enzymes are known for theirthermostability, see WO 2007/091231. The produced mash is used in afermentation during which the reducing sugars are converted into yeastbiomass (cells), carbon dioxide and ethanol. The ethanol produced inthis way is called bio-ethanol.

The common production of sugars from pretreated lignocelullosicfeedstock, the hydrolysis also called liquefaction, presaccharificationor saccharification, typically takes place during a process lasting 6 to168 hours (Kumar, S. Chem. Eng. Technol. 32 (2009) 517-526) underelevated temperatures of 45 to 50° C. and non-sterile conditions. Duringthis hydrolysis, the cellulose present is partly (typically 30 to 95%,dependable on enzyme activity and hydrolysis conditions) converted intoreducing sugars. In case of inhibition of enzymes by compounds presentin the pretreated feedstock and by released sugars and to minimizethermal inactivation, this period of elevated temperature is minimizedas much as possible.

The fermentation following the hydrolysis takes place in a separatepreferably anaerobic process step, either in the same or in a differentvessel, in which temperature is adjusted to 30 to 33° C. (mesophilicprocess) to accommodate growth and ethanol production by microbialbiomass, commonly yeasts. During this fermentation process, theremaining (hemi)cellulosic material is converted into reducing sugars bythe enzymes already present from the hydrolysis step, while microbialbiomass and ethanol are produced. The fermentation is finished once(hemi)cellulosic material is converted into fermentable sugars and allfermentable sugars are converted into ethanol, carbon dioxide andmicrobial cells. This may take up to 6 days. In general, the overallprocess time of hydrolysis and fermentation may amount up to 13 days.

The so-obtained fermented mash consists of non-fermentable sugars,non-hydrolysable (hemi)cellulosic material, lignin, microbial cells(most common yeast cells), water, ethanol, dissolved carbon dioxide.During the successive steps, ethanol is distilled from the mash andfurther purified. The remaining solid suspension is dried and used as,for instance, burning fuel, fertilizer or cattle feed.

WO 2010/080407 suggests treating cellulosic material with a cellulasecomposition under anaerobic conditions. Removal or exclusion of reactiveoxygen species may improve the performance of cellulose hydrolyzingenzyme systems. Hydrolysis of cellulosic material, e.g. lignocellulose,by an enzyme composition can be reduced by oxidative damage tocomponents of the enzyme composition and/or oxidation of the cellulosicmaterial by, for example, molecular oxygen.

WO 2009/046538 discloses a method for treating lignocellulosic feedstockplant materials to release fermentable sugars using an enzymatichydrolysis process for treating the materials performed under vacuum andproducing a sugar rich process stream comprising reduced amounts ofvolatile sugar/fermentation inhibiting compounds such as furfural andacetic acid. Apart from removing volatile inhibitory compounds, othercompounds and/or molecules that are also removed include nitrogen,oxygen, argon and carbon dioxide.

With each batch of feedstock, enzymes are added to maximize the yieldand rate of fermentable sugars released from the pretreatedlignocellulosic feedstock during the given process time. In general,costs for enzymes production, feedstock to ethanol yields andinvestments are major cost factors in the overall production costs(Kumar, S. Chem. Eng. Technol. 32 (2009) 517-526). Thus far, cost ofenzyme usage reduction is achieved by applying enzyme products from asingle or from multiple microbial sources (WO 2008/008793) with broaderand/or higher (specific) hydrolytic activity which use aims at a lowerenzyme need, faster conversion rates and/or a higher conversion yields,and thus at overall lower bioethanol production costs. This requireslarge investments in research and development of these enzyme products.In case of an enzyme product composed of enzymes from multiple microbialsources, large capital investments are needed for production of eachsingle enzyme compound.

It is therefore desirable to improve the above process involvinghydrolysis and fermentation.

SUMMARY OF THE INVENTION

An object of the invention is therefore to provide an apparatus and aprocess in which the hydrolysis step is conducted at improvedconditions. Another object of the invention is to provide an apparatusand a process involving hydrolysis having a reduced process time.Further object of the invention is to provide an apparatus and aprocess, wherein the dosage of enzyme may be reduced and at the sametime output of useful hydrolysis product is maintained at the same levelor even increased. Another object is to provide an apparatus and aprocess involving hydrolysis, wherein the process conditions of thehydrolysis are optimized. A still further object of the invention is toprovide an apparatus and a process involving hydrolysis, wherein theoutput of useful hydrolysis product is increased using the same enzymedosage. One or more of these objects are attained according to theinvention.

The present invention provides a process for the preparation of a sugarproduct from lignocellulosic material, comprising the following steps:

-   -   a) optionally, pretreatment of the lignocellulosic material;    -   b) optionally, washing of the optionally pretreated        lignocellulosic material;    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pretreated lignocellulosic material in a hydrolysis        reactor using an enzyme composition comprising at least two        cellulases; and    -   d) optionally, recovery of a sugar product;        wherein during the enzymatic hydrolysis oxygen-containing gas is        added to the lignocellulosic material in the hydrolysis reactor        and wherein part of the oxygen-containing gas, added to the        lignocellulosic material, is gas originating from the headspace        of the reactor. Preferably, during part of the time of the        enzymatic hydrolysis less oxygen is added to the lignocellulosic        material compared to the other part of the time of the enzymatic        hydrolysis, or preferably during part of the time of the        enzymatic hydrolysis no oxygen is added to the lignocellulosic        material.

Furthermore the present invention provides a process for the preparationof a fermentation product from lignocellulosic material, comprising thefollowing steps:

-   -   a) optionally, pretreatment of the lignocellulosic material;    -   b) optionally, washing of the optionally pretreated        lignocellulosic material;    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pretreated lignocellulosic material in a hydrolysis        reactor using an enzyme composition comprising at least two        cellulases;    -   d) fermentation of the hydrolysed lignocellulosic material to        produce a fermentation product; and    -   e) optionally, recovery of a fermentation product;        wherein during the enzymatic hydrolysis oxygen-containing gas is        added to the lignocellulosic material in the hydrolysis reactor        and wherein part of the oxygen-containing gas, added to the        lignocellulosic material, is gas originating from the headspace        of the reactor. Preferably during part of the time of the        enzymatic hydrolysis less oxygen is added to the lignocellulosic        material compared to the other part of the time of the enzymatic        hydrolysis, or preferably during part of the time of the        enzymatic hydrolysis no oxygen is added to the lignocellulosic        material.

According to a further aspect of the invention an apparatus is providedwhich is suitable for the enzymatic hydrolysis process of the invention.The apparatus according to the invention comprises:

a) a reactor or reactor vessel of at least 1 m³ which comprises a gasintroducing means, preferably a gas sparger, for introduction of gas inthe reactor;b) optionally, a stirring means for stirring the reactor contents;c) a gas pump for introducing gas into the reactor;d) a recycle pipe for recycling gas from the headspace of the reactor;e) an exhaust for deleting gas from the reactor;f) a gas inlet for introducing fresh gas in the reactor;g) a means, preferably a valve, for controlling the ratio betweenrecycled gas and fresh gas.

In general, this apparatus is very suitable for the introduction of agas such as air into a liquid reaction mixture such as the enzymatichydrolysis of biomass having preferably a dry matter content in thehydrolysis step of 10 wt % or more, preferably of 14 wt % or more andstill more preferably of 14 to 33 wt %.

According to a preferred embodiment of the invention the part of thetime wherein less or preferably no oxygen is added is 10 to 80%,preferably 20 to 80%, more preferably 30 to 80% and most preferably 40to 80% of the total enzymatic hydrolysis time.

According to another preferred embodiment of the invention the part ofthe time wherein more oxygen is added is 2 to 80%, preferably 4 to 60%,more preferably 8 to 50% and most preferably 10 to 50% of the totalenzymatic hydrolysis time. More preferably, the part of the time whereinmore oxygen is added is

-   -   a) 12 to 50%, and preferably 20 to 40% when the oxygen is added        in the second half of time of the enzymatic hydrolysis;    -   b) 2 to 30%, preferably 4 to 25% and more preferably 5 to 20% of        the total enzymatic hydrolysis time when the oxygen is added in        the first half of time of the enzymatic hydrolysis; or    -   c) a combination of a and b.

Advantageously, the oxygen concentration in the liquid phase of thehydrolysis during the part of the time wherein oxygen is added is atleast 2 times, preferably at least 4 times, more preferably at least 10times the oxygen concentration in the liquid phase during the part ofthe time wherein less or no oxygen is added.

In an embodiment the oxygen concentration in the liquid phase of thehydrolysis during the total enzymatic hydrolysis time is low.

According to a further preferred embodiment of the invention, in thepart of the time when the oxygen is added, the oxygen concentration inthe liquid phase, wherein the lignocellulosic material is present duringthe enzymatic hydrolysis, is at least 0.001 mol/m³, preferably at least0.002 mol/m³ and most preferably at least 0.003 mol/m³ and even morepreferably more than 0.01 mol/m³, for example more than 0.02 mol/m³ or0.03 mol/m³. In reactors of less than 1 m³, DO values of below 0.01mol/m³ or 0.02 mol/m³ will be obtained by slow stirring. Vigorous mixingor stirring at such scale introduces part of the gas phase of theheadspace into the reaction liquid. For example, the mixing or stirringmay create a whirlpool that draws oxygen into the liquid. In general,flushing the headspace with oxygen (for example in the form of air) incombination with (vigorous) mixing or stirring will introduce sufficientoxygen into the cellulosic material in the hydrolysis reactor forreactors up to a size of 100 liter to 1 m³. At larger scale, for examplein a reactor of 50 m³ or more, for example 100 m³, so much energy isneeded for vigorous stirring that from an economic point of view thiswill not be applied in a commercially operating process. In general, inlarge reactors stirring or mixing without introducing air or oxygen willresult in DO values of less than 0.01 mol/m³.

To still another preferred embodiment of the invention during the oxygenaddition (in the part of the time when the oxygen is added), the oxygenconcentration in the liquid phase, wherein the lignocellulosic materialis present during the enzymatic hydrolysis, is preferably at most 80% ofthe saturation concentration of oxygen under the hydrolysis reactionconditions, more preferably at most 0.12 mol/m³, still more preferablyat most 0.09 mol/m³, even more preferably at most 0.06 mol/m³, evenstill more preferably at most 0.045 mol/m³ and most preferably at most0.03 mol/m³. In an embodiment the oxygen concentration is 0 mol/m³,since the oxygen consumption is higher than the oxygen transfer rate.Temperature and pressure will influence the DO. The preferred andexemplary mol/m³ values given above relate to normal atmosphericpressure and a temperature of about 62° C. The skilled person in the artwill appreciate favourable DO values on basis of the present teachings.

According to a further preferred embodiment of the invention oxygen isconsumed in an amount corresponding to between 20 and 5000 mmolmolecular oxygen per kg glucan present in the lignocellulosic material.Preferably, oxygen is consumed in an amount corresponding to between 22and 4500 mmol molecular oxygen per kg glucan present in thelignocellulosic material, between 24 and 4000 mmol molecular oxygen perkg glucan present in the lignocellulosic material, between 26 and 3500mmol molecular oxygen per kg glucan present in the lignocellulosicmaterial, between 28 and 3000 mmol molecular oxygen per kg glucanpresent in the lignocellulosic material. The oxygen is added after thepretreatment and before and/or during the enzymatic hydrolysis of thelignocellulosic material, preferably in an amount corresponding to atleast 30 mmol molecular oxygen per kg glucan present in thelignocellulosic material, more preferably in an amount corresponding toat least 40 mmol molecular oxygen per kg glucan present in thelignocellulosic material, and most preferably in an amount correspondingto at least 50 mmol molecular oxygen per kg glucan present in thelignocellulosic material. All oxygen that is added to the system will betransferred to the liquid and used for the hydrolysis. This amount canbe controlled by measuring and controlling the amount of air broughtinto the system.

According to another preferred embodiment of the invention the reactorfor the enzymatic hydrolysis has a volume of 1 m³ or more. Preferably,the reactor has a volume of at least 1 m³, at least 2 m³, at least 3 m³,at least 4 m³, at least 5 m³, at least 6 m³, at least 7 m³, at least 8m³, at least 9 m³, at least 10 m³, at least 15 m³, at least 20 m³, atleast 25 m³, at least 30 m³, at least 35 m³, at least 40 m³, at least 45m³, at least 50 m³, at least 60 m³, at least 70 m³, at least 75 m³, atleast 80 m³, at least 90 m³, at least 100 m³, at least 200 m³, at least300 m³, at least 400 m³, at least 500 m³, at least 600 m³, at least 700m³, at least 800 m³, at least 900 m³, at least 1000 m³, at least 1500m³, at least 2000 m³, at least 2500 m³. In general, the reactor will besmaller than 3000 m³ or 5000 m³. Several reactors may be used. Thereactors used in the processes of the present invention may have thesame volume, but also may have a different volume. The enzymatichydrolysis time of the present process is preferably from 5 to 150hours.

According to a further preferred aspect of the invention the enzymecomposition is derived from a fungus, preferably a microorganism of thegenus Rasamsonia or the enzyme composition comprises a fungal enzyme,preferably a Rasamsonia enzyme. According to a still further preferredaspect of the invention the dry matter content in the hydrolysis step c)is 10 wt % or more, preferably is 14 wt % or more and still morepreferably is 14 to 33 wt %. The enzymatic hydrolysis preferably takesplace in a batch, fed batch and/or continuous culture reactor.Preferably, the oxygen that is introduced in the present process is anoxygen-containing gas such as air. By less oxygen is added to or ispresent in the lignocellulosic material during part of the time of theenzymatic hydrolysis, is meant that at least 50% less, preferably atleast 70% less, most preferably at least 90% less of oxygen (expressedin mol oxygen/m³) is introduced, for example in bubble form or ispresent than is added or is present during the other part of the time ofthe enzymatic hydrolysis wherein oxygen is added.

In a preferred embodiment the oxygen is added in the form of (gaseous)bubbles.

Surprisingly, according to the invention, by the addition of oxygen itis possible to attain many process advantages, including optimaltemperature conditions, reduced process time, reduced dosage of enzyme,re-use of enzymes, higher yields and other process optimizations,resulting in reduced costs.

In an embodiment the stable enzyme composition used retains activity for30 hours or more. According to a further embodiment the hydrolysis ispreferably conducted at a temperature of 40° C. or more, more preferablyat a temperature of 50° C. or more and most preferably at a temperatureof 55° C. or more. The process of the invention will be illustrated inmore detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The effect of sparging nitrogen or air through a 10% aCSfeedstock before hydrolysis, on the total amount of glucose (g/1)released by the TEC-210 mix.

FIG. 2: The glucose produced in Example 2, 1=Experiment 1: no aeration,2=Experiment 2: continuous aeration, 3=Experiment 3: aeration startingat 72 hours until the end.

FIG. 3: The effect of time of aeration on glucose produced duringenzymatic hydrolysis, — —=no aeration, ●●●=aeration between hydrolysistime is 0 and 100 hours, - - - aeration between hydrolysis time is 0 and7 hours and — — —=aeration between hydrolysis time is 72 and 100 hours.

FIG. 4: The effect of time of aeration on glucose produced duringenzymatic hydrolysis in experiment 1 (▪=aeration between hydrolysis timeis 0 and 100 hours) and 2 (□=aeration between hydrolysis time is 72 and100 hours).

FIG. 5: The effect of time of aeration on glucose produced duringenzymatic hydrolysis, —▪— aeration between hydrolysis time is 72 and 100hours and —●— aeration between hydrolysis-time is 0 and 7 hours.

FIG. 6: A schematic design for an embodiment of the apparatus of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows. The articles “a” and “an” are used herein to referto one or to more than one (i.e. to one or at least one) of thegrammatical object of the article. By way of example, “an element” maymean one element or more than one element.

In the context of the present invention “improved”, “increased”,“reduced” is used to indicate that the present invention shows anadvantage compared to the same situation, process or process conditionsexcept that no extra oxygen is added. Within the context of the presentinvention “measured under the same conditions” or “analysed under thesame conditions” etc. means that the process of the invention and thesame process without (or with less) addition of oxygen are performedunder the same conditions (except the oxygen addition) and that theresults of the present process, if compared to the process without (orwith less) oxygen addition, are measured using the same conditions,preferably by using the same assay and/or methodology, more preferablywithin the same or parallel experiment. Conditions of the hydrolysis arean example of such conditions.

In prior art it is suggested to improve the hydrolysis of cellulolyticmaterial by using anaerobic (WO 2010/080407) or vacuum (WO 2009/046538)conditions during the enzymatic hydrolysis. In the processes of bothdocuments the oxygen level was decreased. It has been surprisingly foundthat the hydrolysis of the present invention shows results in animproved reaction product that gives higher amounts of (reduced) sugarproducts and/or desired fermentation products in the fermentationfollowing the hydrolysis as compared to a process wherein no oxygen isadded. In general, an increase of the glucose conversion is observed of5 to 15 w/w %, or even up to 25 w/w %.

Oxygen can be added in several ways. For example, oxygen can be added asoxygen gas, oxygen-enriched gas such as oxygen-enriched air or air(example of oxygen containing gas). Oxygen can be added continuously ordiscontinuously. By oxygen “is added” is meant that oxygen is added tothe liquid phase (comprising the lignocellulosic material) in thehydrolysis reactor and not that oxygen is present in the headspace inthe reactor above the liquid phase (in combination with slow or nostirring) whereby the oxygen has to diffuse from the headspace to theliquid phase. So preferably, the oxygen is added as bubbles, mostpreferably as small bubbles. In an embodiment the bubbles have adiameter of at least 0.5 mm, at least 1 mm, at least 1.5 mm, at least 2mm, at least 2.5 mm, at least 3 mm, at least 3.5 mm, at least 4 mm, atleast 4.5 mm, at least 5 mm. In an embodiment the bubbles have adiameter of between 0.5 mm and 500 mm, preferably between 0.5 and 400mm, between 0.5 and 300 mm, between 0.5 and 200 mm, between 0.5 and 100mm.

In case the enzyme may be damaged by the presence or addition of oxygen,milder oxygen supply may be used. In that case, a balance can be foundbetween the improved glucose production and the enzyme performance. Theaddition of the oxygen to the cellulolytic material can be done duringthe enzymatic hydrolysis. In case oxygen is added in gaseous form,oxygen-containing gas can be introduced, for example blown, into theliquid hydrolysis reactor contents of cellulolytic material. In anotherembodiment of invention the oxygen-containing gas is introduced into theliquid cellulolytic material stream that will enter the hydrolysisreactor. In still another embodiment of the invention the oxygencontaining gas is introduced together with the cellulolytic materialthat enters the hydrolysis reactor or with part of the liquid reactorcontents that passes an external loop of the reactor. In most cases, theaddition of oxygen before entering the hydrolysis reactor is notsufficient enough and oxygen addition may be done during the hydrolysisas well. In another embodiment of the invention the gaseous phasepresent in the upper part of the reactor (head space) is continuously ordiscontinuously refreshed with the oxygen-containing gas. In the lattercase (vigorous) mixing or stirring is needed to get the oxygen asbubbles and/or by diffusion into the liquid reactor contents preferablyin combination with overpressure in the reactor. In general, flushingthe headspace with air in combination with (vigorous) mixing or stirringmay introduce sufficient oxygen into the cellulosic material in thehydrolysis reactor for reactors up to a size of 100 liter to 1 m³. Atlarger scale, for example in a reactor of 50 m³ or more, for example 100m³, so much energy is needed for vigorous stirring that from economicpoint of view this will not be applied in a commercially operatingprocess.

According to the present invention the oxygen may be added during partof the hydrolysis step. The addition of oxygen during only part of thehydrolysis may be done for example in case of oxidation damage of theenzyme(s) occurs. In case the oxygen present in the hydrolysis reactorcontents or the sugar product or hydrolysate formed in the hydrolysisstep might influence or disturb the subsequent fermentation step, oxygenaddition may be done except for the last part of the hydrolysis and thus(most of) the oxygen is consumed before the hydrolyzed biomass entersthe fermentation reactor. Advantageously, the oxygen, preferably in theform of (gaseous) bubbles, is added in the last part of the hydrolysisstep.

The inventors pose the hypothesis that in the first part of the(enzymatic) hydrolysis (step) amorphous polysaccharides are hydrolysedto sugars such as glucose and that in the second part of the hydrolysisstep the remaining crystalline polysaccharides are converted to sugars.Amorphous polysaccharides are for example converted to oligosaccharidesby endogluconases, whereafter the oligosaccharides can be converted bycellobiohydrolase and beta-glucosidase (BG) to sugars. According to thepresent hypothesis amorphous polysaccharides are located on the outsideof polysaccharides or polysaccharide complexes, whereas crystallinepolysaccharides are located relatively more in the inside of thepolysaccharides or polysaccharide complexes present in thelignocellulosic material. So, the conversion of the crystallinepolysaccharides may continue even when most of the amorphouspolypeptides are hydrolysed. Especially, the addition of oxygen isbeneficial during the hydrolysis of the crystalline polysaccharides, forexample in the degradation of the polysaccharides into oligosaccharides.According to this hypothesis oxygen addition is especially useful in thesecond part of the hydrolysis step. In general, a shorter time of oxygenaddition (or shorter second part of hydrolysis) is needed in case ofrelatively low amounts of crystalline polysaccharides in thelignocellulosic material compared hydrolysis of lignocellulosic materialin which relatively higher amounts of crystalline polysaccharides arepresent. The inventors also pose that the addition of oxygen isbeneficial for the hydrolysis of crystalline polysaccharides. Therefore,the addition of oxygen is very useful especially in the phase whereincrystalline polysaccharides are attacked by enzymes. Outside this phasenot adding of oxygen might be more efficient. Therefore, the oxygensupply may start only in the second part or second half of thehydrolysis. At the end of the hydrolysis, when most of the crystallinepolysaccharides are degraded, the oxygen addition is preferably stopped.In the last part of the second part or second half of the hydrolysismost of the polysaccharides are converted to oligosaccharides whichduring further breakdown to smaller sugars do not need oxygen anymore.Therefore, preferably less oxygen, compared to the oxygen additionduring the aerated part of the time, is added to the lignocellulosicmaterial at the end of the hydrolysis process or more preferably nooxygen is added to the lignocellulosic material at the end of thehydrolysis process. This hypothesis is only given as possibleexplanation of the effect noticed by the inventors and the presentinvention does not fall or stand with the correctness of this theory.

The crystalline glucan structure can be opened by a lytic polysaccharidemonooxygenase (LPMO). LPMOs are described in more detail below (they arealso called PMOs). This type of enzyme opens up the structure byoxidizing the glycosidic bonds and making it accessible for the othercellulolytic enzymes for further hydrolysing the oligosaccharides intoglucose.

Most known LPMO's form aldonic acids, i.e. products oxidized at the C1position of the terminal sugar at the cleavage site. This oxidizedglucose unit is released as gluconic acid during hydrolysis. Inaddition, oxidation of the C4 and C6 of the non-reducing glucose unit atthe cleavage site has been reported. For instance, T. Isaksen et. al.(vide supra) reported the oxidation of the C4 position, the non-reducingend moiety, resulting in a keto-sugar at the C4 position, which is inequilibrium with a C4 geminal diol in water solution. The presentinventors pose that hydrolysed oxidation products, like for examplegluconic acid, are a measure for the performance of the applied LPMO inlignocellulose hydrolysis.

Surprisingly, the present inventors have found that optimallignocellulose hydrolysis (more than 70% glucan conversion) can beobtained by oxygen consumption of an amount corresponding to between 20and 5000 mmol molecular oxygen per kg glucan present in thelignocellulosic material after the pretreatment and before and/or duringthe enzymatic hydrolysis to the lignocellulosic material, preferably inan amount corresponding to at least 30 mmol molecular oxygen per kgglucan present in the lignocellulosic material, more preferably in anamount corresponding to at least 40 mmol molecular oxygen per kg glucanpresent in the lignocellulosic material, and most preferably in anamount corresponding to at least 50 mmol molecular oxygen per kg glucanpresent in the lignocellulosic material. According to a furtherpreferred embodiment of the invention oxygen is consumed in an amountcorresponding to between 20 and 5000 mmol molecular oxygen per kg glucanpresent in the lignocellulosic material. Preferably, oxygen is consumedin an amount corresponding to between 22 and 4500 mmol molecular oxygenper kg glucan present in the lignocellulosic material, between 24 and4000 mmol molecular oxygen per kg glucan present in the lignocellulosicmaterial, between 26 and 3500 mmol molecular oxygen per kg glucanpresent in the lignocellulosic material, between 28 and 3000 mmolmolecular oxygen per kg glucan present in the lignocellulosic material.The oxygen is added after the pretreatment and before and/or during theenzymatic hydrolysis of the lignocellulosic material, preferably in anamount corresponding to at least 30 mmol molecular oxygen per kg glucanpresent in the lignocellulosic material, more preferably in an amountcorresponding to at least 40 mmol molecular oxygen per kg glucan presentin the lignocellulosic material, and most preferably in an amountcorresponding to at least 50 mmol molecular oxygen per kg glucan presentin the lignocellulosic material. All oxygen that is added to the systemwill be transferred to the liquid and used for the hydrolysis. Thisamount can be controlled by measuring and controlling the amount of airbrought into the system.

This amount can be controlled by measuring and controlling the amount ofair brought into the system.

Oxidation by LPMO of the lignocellulosic material results in oxidisedpolysaccharides which during the hydrolysis are hydrolysed into amongstothers in glucose and oxidised glucose units such as gluconic acid ordiol. In general 1 molecule oxygen (O₂) gives one mol oxidation product.It will be evident that optimal lignocellulose hydrolysis can only beachieved when (crystalline) cellulose and cello-oligosaccharides arehydrolysed optimally. This optimal hydrolysis by the action of a LPMOwill result in the formation of hydrolysed oxidation product, likegluconic acid. No oxidation means a less efficient hydrolysis of(crystalline) glucan. However, too high levels of oxidation will resultin higher levels of products like gluconic acid and will be at theexpense of glucose and therefore the glucose yield on (starting) glucanwill go down.

The inventors have also noticed that aeration during an enzymatichydrolysis process in the beginning of the hydrolysis process results inan increased glucose production during the hydrolysis.

In FIG. 3 the effect of aeration is shown. Compared to the non-aeratedhydrolysis (shown as “non-aerated” curve), an aeration at the start ofthe hydrolysis process (shown as “aeration 0-7 hours” curve) will resultin an immediate increase in glucose production and for example alreadyafter 24 hours of hydrolysis a glucose production will be found thatcorresponds to a glucose production without aeration of 60 hourshydrolysis under identical conditions (except for aeration). Compared tothe non-aerated hydrolysis, an aeration at the last part of thehydrolysis process (shown as “aeration 72-100 hours” curve) will resultin an immediate increase in glucose production after aeration and forexample already after 24 hours after the start of aeration (at 72 hours)in the hydrolysis process a glucose production increase of 30% will befound compared to the glucose production without aeration underidentical conditions (except for aeration). It is believed by theinventors that by using an aeration at the start as well as at the lastpart of the hydrolysis process (with in between the aeration intervals aperiod of no aeration) might increase glucose production, whereby thisresults in an increase of glucose production that is larger than one ofthe two separate increases. The present explanation is given to guideand instruct the skilled person in the art to select the properconditions for the present hydrolysis process.

Several examples of partial aeration during the enzymatic hydrolysisprocess are given in the Examples to show the beneficial effect of thepresent invention. This beneficial effect is found for severalsubstrates or feedstocks and therefore believed to be present for thehydrolysis of all kind of substrates or feedstocks.

Several examples of enzyme compositions for the enzymatic hydrolysisprocess are given in the Examples to show the beneficial effect of thepresent invention. This beneficial effect is found for several enzymecompositions and therefore believed to be present for all kind ofhydrolysing enzyme compositions.

According to a preferred embodiment of the invention the part of thetime wherein less or preferably no oxygen is added is 10 to 80%,preferably 20 to 80%, more preferably 30 to 80% and most preferably 40to 80% of the total enzymatic hydrolysis time. According to a furtherpreferred embodiment of the invention the part of the time wherein moreoxygen is added is 2 to 80%, preferably 4 to 60%, more preferably 8 to50% and most preferably 10 to 50% of the total enzymatic hydrolysistime. In general, the oxygen concentration in the liquid phase duringthe part of the time wherein oxygen is added is at least 2 times,preferably at least 4 times, more preferably at least 10 times theoxygen concentration in the liquid phase during the part of the timewherein less or no oxygen is added.

To a further preferred embodiment of the invention during the part ofthe time wherein oxygen addition takes place in the liquid phase (byaeration or addition of oxygen), the oxygen concentration (DO) in theliquid phase, wherein the lignocellulosic material is present during theenzymatic hydrolysis, is at least 0.001 mol/m³, preferably at least0.002 mol/m³, more preferably at least 0.003 mol/m³ and even morepreferably more than 0.01 mol/m³, for example more than 0.02 mol/m³ or0.03 mol/m³. In reactors of less than 1 m³ DO values of below 0.01mol/m³ or 0.02 mol/m³ will be obtained by slow stirring. Vigorous mixingor stirring at such scale introduces part of the gas phase of theheadspace into the reaction liquid. For example, the mixing or stirringmay create a whirlpool that draws oxygen into the liquid. In general,flushing the headspace with air in combination with (vigorous) mixing orstirring will introduce sufficient oxygen into the cellulosic materialin the hydrolysis reactor for reactors up to a size of 100 liter to 1m³. At larger scale, for example in a reactor of 50 m³ or more, forexample 100 m³, so much energy is needed for vigorous stirring that fromeconomic point of view this will not be applied in a commerciallyoperating process. In general, in large reactors, stirring or mixingwithout introducing air or oxygen will result in DO values of less than0.01 mol/m³.

To still another preferred embodiment of the invention during the oxygengeneration or production the oxygen concentration in the liquid phase(aeration or addition of oxygen), the oxygen concentration in the liquidphase, wherein the lignocellulosic material is present during theenzymatic hydrolysis, is during the part of the time wherein oxygen isadded preferably at most 80% of the saturation concentration of oxygenunder the hydrolysis reaction conditions, more preferably at most 0.12mol/m³, still more preferably at most 0.09 mol/m³, even more preferablyat most 0.06 mol/m³, even still more preferably at most 0.045 mol/m³ andmost preferably at most 0.03 mol/m³. In an embodiment the oxygenconcentration is 0 mol/m³, since the oxygen consumption is higher thanthe oxygen transfer rate. Temperature and pressure will influence theDO. The preferred and exemplary mol/m³ values given above relate tonormal atmospheric pressure and a temperature of about 62° C. Theskilled person in the art will appreciate favourable DO values on thebasis of the present teachings.

To a further preferred embodiment of the invention the oxygenconcentration in the liquid phase, wherein the lignocellulosic materialis present during the enzymatic hydrolysis, is during the part of thetime wherein less or no oxygen is added less than 0.02 mol/m³,preferably less than 0.01 mol/m³, more preferably less than 0.005mol/m³, and most preferably less than 0.001 mol/m³.

The oxygen addition in the form of air or other oxygen-containing gasaccording to the invention may also be used to at least partially stiror mix the hydrolysis reactor contents. The present process of theinvention shows especially on pilot plant and industrial scaleadvantages. Preferably, the hydrolysis reactor has a volume of 1 m³ ormore, preferably of more than 10 m³ and most preferably of 50 m³ ormore. In general the hydrolysis reactor will be smaller than 3000 m³ or5000 m³. The inventors pose the theory that especially at large scaleinsufficient oxygen is available for the hydrolysis which might be dueto oxygen transfer limitations in the reactor for example in thecellulolytic biomass. On lab-scale experiments this oxygen-insufficiencymay play a less important role. The surface area (or oxygen contact areaof the reactor content) to reactor volume ratio is more favourable forsmall scale experiments than in large scale experiments. Moreover,mixing in small scale experiments is relatively easier than at largescale. During those small scale experiments also the transport of oxygenfrom the headspace of the hydrolysis reactor is faster than compared tothe situation in large scale experiments. This theory is only given aspossible explanation of the effect noticed by the inventors, and thepresent invention does not fall or stands with the correctness of thistheory. According to a further embodiment of the invention the additionof oxygen may be used to control at least partially the hydrolysisprocess.

According to a further aspect of the invention an apparatus is providedsuitable for the enzymatic hydrolysis of the invention. The apparatusaccording to the invention comprises:

a) a reactor or reactor vessel of at least 1 m³ which comprises a gasintroducing means, preferably a gas sparger for introduction of gas inthe reactor;b) optionally, a stirring means for stirring the reactor contents;c) a gas pump for introducing gas into the reactor;d) a recycle pipe for recycling gas from the headspace of the reactor;e) an exhaust for deleting gas from the reactor;f) a gas inlet for introducing fresh gas in the reactor;g) a means, preferably a valve, for controlling the ratio betweenrecycled gas and fresh gas.

In FIG. 6 one embodiment of the apparatus of the invention is givenschematically.

Preferably, the process of the invention is performed in the apparatusof the present invention. Advantageously, during at least part of thetime the process of the present invention is performed in the presentapparatus and the gas introduced in the reactor is oxygen-containinggas. The gas introduced in the gas inlet is preferably oxygen-containinggas and more preferably is air. In the process of the invention oxygenis consumed and the recycled gas will contain less oxygen than the gasintroduced in the reactor. By the means, preferably a valve, forcontrolling the ratio between recycled gas and fresh gas, the oxygen canbe introduced in the reactor in exactly the desired amount needed forthe enzymatic hydrolysis reaction. The oxygen level in the gasintroduced in the reactor can be controlled between the level of oxygenin the headspace and the oxygen level in the gas inlet. In case no gasis introduced by the gas inlet, only recycled gas will be introduced inthe reactor. The oxygen consumption during the enzymatic hydrolysis willresult in the latter case in a decrease in the oxygen content present inthe apparatus until finally oxygen is completely absent.

In case hardly any gas is recycled compared to freshly introduced gasvia the gas inlet, the oxygen level of the gas present in the apparatuswill be close to the oxygen level of the freshly introduced gas. In casethe freshly introduced gas is air, the oxygen level in the reactor canbe advantageously controlled by selecting the proper ratio betweenfreshly introduced air and recycled gas.

Maintaining the oxygen level at a selected value can be obtained byintroducing the same amount of fresh oxygen through the gas inlet as theamount of oxygen that is consumed during the hydrolysis in the reactor,

-   -   The gas sparger makes introduction of gas possible in the        reactor. A gas pump will provide the necessary pressure to        introduce the gas in the reactor through the sparger. By        selecting the pressure, the amount of gas introduced through the        sparger can be controlled. Gas can be used for the introduction        of oxygen in the reactor as well as for the mixing of the        reactor contents in the reactor. The gas sparger may have for        example nozzles or orifices for the introduction of the gas into        the reactor content. In general, the diameter of the holes in        the sparger will be at least 0.5 mm, at least 1 mm, at least 1.5        mm, at least 2 mm, at least 2.5 mm, at least 3 mm, at least 3.5        mm, at least 4 mm, at least 4.5 mm, at least 5 mm. In an        embodiment diameter of the holes in the sparger are between 0.5        mm and 500 mm, preferably between 0.5 and 400 mm, between 0.5        and 300 mm, between 0.5 and 200 mm, between 0.5 and 100 mm.

Optionally, a stirrer can be used for mixing the reactor contents aswell. In case of the use of a stirrer in the reactor, all stirrers knownin the art can be used for example anchor-type, peddle-type,propeller-type etc. In case of use of a stirrer one other more stirrerscan be used. Each impeller may have for example 2, 3, 4 or more blades,the blades of the impeller of the stirrer may be vertical blades orpitched blades, vertical and pitched with respect to the axis of thestirrer.

Optionally, a (circulating) liquid stream can be used to drive a venturetube for the introduction of oxygen containing gas in the liquid. Thiscirculate liquid stream system will circulate (part of) the liquidreactor contents namely the hydrolysate or lignocellulosic material.

In general, cellulase compositions that are used for the hydrolysis oflignocellulosic material will comprise several cellulases includingGH61. GH61 is capable of hydrolysing crystalline cellulose intocellulose polymers which can be further hydrolysed into glucose by othercellulases. Oxygen is needed for the hydrolysis reaction catalysed byGH61 and consequently the glucose unit on the reducing end of thehydrolyse cellulose polymer is oxidized into a gluconic acid residue.This gluconic acid residue is liberated when hydrolysis progresses. Itwill be evident that limited GH61 activity may result in only partialhydrolysis of crystalline cellulose, while on the other hand excessivehydrolysis will lead to high gluconic acid concentrations and reducedglucose liberation from the cellulosic feedstock. Therefore, the actionof GH61 is advantageously controlled for an optimal result. One way ofsupplying oxygen during the hydrolysis is adding oxygen in the form ofan oxygen-containing gas such as air. According to the present inventionthe oxygen added and present, and thus the oxygen level, in thehydrolysis is accurately controlled by the apparatus described herein.

According to the present invention the control of the amount of oxygensupplied to the hydrolysate is of paramount importance. In addition,local high concentrations of oxygen need to be avoided in thelignocellulosic material (hydrolysate) in the hydrolysis reactor inorder to limit enzyme activity loss due to the effect of oxygen oncellulases. This is done by making sure that the liquid is properlymixed and the oxygen is dosed and controlled at a desired oxygenconcentration in the reactor. By circulating the gas from the top of thevessel (head space of the reactor) to a gas introduction system such asa sparger at the bottom, all oxygen present in the head space at thestart is consumed very quickly. This consumption occurs amongst othersdue to the oxidation of the present lignin and the action of GH61. Afterthe moment that the oxygen is consumed, the gas above the hydrolysatemainly contains nitrogen and carbon dioxide. The recirculation ofexhausted gas (containing hardly any or no oxygen anymore) can providesufficient mixing in the reactor in case the gas is recirculated througha gas introduction device such as a sparger that “covers” a large partof the bottom and contains small holes to provide small bubbles. By“covers” is meant that the small gas bubbles are produced and arepresent over large part or portion of the reactor contents at the bottomand thus not only at a limited portion of the bottom contents. The sizeof the holes is preferably below 1 cm in diameter.

If a small amount of oxygen for example in the form of air is suppliedinto that recycling or recirculation stream, the concentration of oxygenin the recycled air flow will be low. The oxygen supplied into therecycling system can be introduced directly in the gas recycling systemor can be added separately from the recycled gas into the hydrolysate.In this way, the dissolved oxygen concentration in the reactor and theoxygen flux to the liquid in the reactor can be controlled at veryprecise and low levels and can be easily adjusted to the desiredconcentration. The present invention provides a process and apparatuswhich avoids or limits the loss of enzyme activity and at the same timeenables oxygen supply to the hydrolysate.

The process of the invention is advantageously applied in combinationwith the use of thermostable enzymes.

A “thermostable” enzyme means that the enzyme has a temperature optimum60° C. or higher, for example 70° C. or higher, such as 75° C. orhigher, for example 80° C. or higher such as 85° C. or higher. They mayfor example be isolated from thermophilic microorganisms, or may bedesigned by the skilled person and artificially synthesized. In oneembodiment the polynucleotides may be isolated or obtained fromthermophilic or thermotolerant filamentous fungi or isolated fromnon-thermophilic or non-thermotolerant fungi, but are found to bethermostable.

By “thermophilic fungus” is meant a fungus that grows at a temperatureof 50° C. or above. By “themotolerant” fungus is meant a fungus thatgrows at a temperature of 45° C. or above, having a maximum near 50° C.

Examples of thermophilic fungal strains are Rasamsonia emersonii(formerly known as Talaromyces emersoni; Talaromyces emersonii,Penicillium geosmithia emersonii and Rasamsonia emersonii are usedinterchangeably herein).

Suitable thermophilic or thermotolerant fungal cells may be a Humicola,Rhizomucor, Myceliophthora, Rasamsonia, Talaromyces, Thermomyces,Thermoascus or Thielavia cell, preferably a Rasamsonia emersonii cell.Preferred thermophilic or thermotolerant fungi are Humicola grisea var.thermoidea, Humicola lanuginosa, Myceliophthora thermophila, Papulasporathermophilia, Rasamsonia byssochlamydoides, Rasamsonia emersonii,Rasamsonia argillacea, Rasamsonia eburnean, Rasamsonia brevistipitata,Rasamsonia cylindrospora, Rhizomucor pusillus, Rhizomucor miehei,Talaromyces bacillisporus, Talaromyces leycettanus, Talaromycesthermophilus, Thermomyces lenuginosus, Thermoascus crustaceus,Thermoascus thermophilus Thermoascus aurantiacus and Thielaviaterrestris.

Thermophilic fungi are not restricted to a specific taxonomic order andoccur all over the fungal tree of life. Examples are Rhizomucor in theMucorales, Myceliophthora in Sordariales and Talaromyces, Thermomycesand Thermoascus in the Eurotiales (Mouchacca 1997). The majority ofTalaromyces species are rnesophiles but exceptions are species withinsections Emersorii and Thermophila. Section Emersonii includesTalaromyces emersonii, Talaromyces byssochlamydoides, Talaromycesbacillisporus and Talaromyces leycettanus, all of which grow well at 40°C. Talaromyces bacillisporus is thermotolerant, T. leycettanus isthermotolerant to thermophilic, and T. emersonii and T.byssochlamydoides are truly thermophilic (Stolk and Samson 1972). Thesole member of Talaromyces section Thermophila, T. thermophilus, growsrapidly at 50° C. (Evans and Stolk 1971; Evans 1971; Stolk and Samson1972). The current classification of these thermophilic Talaromycesspecies is mainly based on phenotypic and physiological characters, suchas their ability to grow above 40° C., ascospore color, the structure ofascornatal covering and the formation of a certain type of anamorph.Stolk and Samson (1972) stated that the members of the section Emersoniihave anamorphs of either Paecilomyces (T. byssochlamydoides and T.leycettanus) or Penicillium cylindrosporum series (T. emersonii and T.bacillisporus). Later, Pitt (1979) transferred the species belonging tothe Penicillium cylindrosporum series to the genus Geosmithia, based onvarious characters such as the formation of conidia from terminal poresinstead of on collula (necks), a character of Penicillium andPaecilomyces. Within the genus Geosmithia, only G. argillacea isthermotolerant, and Stolk et al. (1969) and Evans (1971) proposed aconnection with members of Talaromyces sect. Emersonii. The phylogeneticrelationship of the themophilic Talaromyces species within Talaromycesand the Trichocomaceae is unknown. See J. Houbraken, Antonie vanLeeuwenhoek 2012 February; 101(2): 403-21.

Rasamsonia is a new genus comprising thermotolerant and thermophilicTalaromyces and Geosmithia species (J. Houbraken et al vida supra).Based on phenotypic, physiological and molecular data, Houbraken et alproposed to transfer the species T. emersonii, T. byssochlamydoides, T.eburneus, G. argillacea and G. cylindrospora to Rasamsonia gen. nov.Talaromyces emersonii, Penicillium geosmithia emersonii and Rasamsoniaemersonii are used interchangeably herein.

Preferred thermophilic fungi are Rasamsonia byssochlamydoides,Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus,Thermoascus crustaceus, Thermoascus thermophilus and Thermoascusaurantiacus.

“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Beauvaria,Cephalosporium, Ceriporiopsis, Chaetomium paecilomyces, Chrysosporium,Claviceps, Cochiobolus, Coprinus, Cryptococcus, Cyathus, Emericella,Endothia, Endothia mucor, Filibasidium, Fusarium, Geosmithia,Gilocladium, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Panerochaete, Pleurotus, Podospora, Pyricularia, Rasamsonia, Rhizomucor,Rhizopus, Scylatidium, Schizophyllum, Stagonospora, Talaromyces,Thermoascus, Thermomyces, Thielavia, Tolypocladium, Trametes pleurotus,Trichoderma and Trichophyton.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). Examples of such strains includeAspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177,ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicilliumchrysogenum P2, Talaromyces emersonii CBS 393.64, Acremonium chrysogenumATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 orATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense C1,Garg 27K, VKM F-3500-D, ATCC44006 and derivatives thereof.

An advantage of expression and production of the enzymes (for example atleast two, three or four different cellulases) in a suitablemicroorganism may be a high enzyme composition yield which can be usedin the process of the present invention.

According to the invention, by the addition of oxygen it is possible toattain many process advantages, including optimal temperatureconditions, reduced process time, reduced dosage of enzyme, re-use ofenzymes and other process optimizations, resulting in reduced costs.Advantageously, the invention provides a process in which the hydrolysisstep is conducted at improved conditions. The invention also provides aprocess involving hydrolysis having a reduced process time. Furthermore,the invention provides a process, wherein the dosage of enzyme may bereduced and at the same time output of useful hydrolysis product ismaintained at the same level. Another advantage of the invention is thatthe present process involving hydrolysis may result in processconditions which are optimized. A still further advantage of theinvention is that the output of useful hydrolysis product of the processinvolving hydrolysis is increased using the same enzyme dosage.

Stable Enzyme Composition

Stable enzyme composition herein means that the enzyme compositionretains activity after 30 hours of hydrolysis reaction time, preferablyat least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% of its initial activity after 30 hours ofhydrolysis reaction time. Preferably, the enzyme composition retainsactivity after 40, 50, 60, 70, 80, 90 100, 150, 200, 250, 300, 350, 400,450, 500 hours of hydrolysis reaction time.

In an embodiment the enzyme composition used in the processes of thepresent invention is derived from a fungus or the enzyme compositionused in the processes of the present invention comprises a fungalenzyme. In an embodiment the enzyme composition is derived from afilamentous fungus or the enzyme composition comprises a filamentousfungal enzyme. The processes of the invention are advantageously appliedin combination with enzyme compositions derived from a microorganism ofthe genus Rasamsonia, or the enzyme composition comprises a Rasamsoniaenzyme.

The enzyme composition may be prepared by fermentation of a suitablesubstrate with a suitable microorganism, e.g. Rasamsonia emersonii orAspergillus niger wherein the enzyme composition is produced by themicroorganism. The microorganism may be altered to improve or to makethe cellulase composition. For example, the microorganism may be mutatedby classical strain improvement procedures or by recombinant DNAtechniques. Therefore, the microorganisms mentioned herein can be usedas such to produce the cellulase composition or may be altered toincrease the production or to produce an altered cellulase compositionwhich might include heterologous cellulases, thus enzymes that are notoriginally produced by that microorganism. Preferably a fungus, morepreferably a filamentous fungus is used to produce the cellulasecomposition. Advantageously, a thermophilic or thermotolerantmicroorganism is used. Optionally, a substrate is used that induces theexpression of the enzymes in the enzyme composition during theproduction of the enzyme composition.

The enzyme composition is used to release sugars from lignocellulosethat comprises polysaccharides. The major polysaccharides are cellulose(glucans), hemicelluloses (xylans, heteroxylans and xyloglucans). Inaddition, some hemicellulose may be present as glucomannans, for examplein wood-derived feedstocks. The enzymatic hydrolysis of thesepolysaccharides to soluble sugars, including both monomers andmultimers, for example glucose, cellobiose, xylose, arabinose,galactose, fructose, mannose, rhamnose, ribose, galacturonic acid,glucoronic acid and other hexoses and pentoses occurs under the actionof different enzymes acting in concert. By sugar product is meant theenzymatic hydrolysis product of the feedstock or lignocellulosicmaterial. The sugar product will comprise soluble sugars, including bothmonomers and multimers, preferably will comprise glucose. Examples ofother sugars are cellobiose, xylose, arabinose, galactose, fructose,mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and otherhexoses and pentoses. The sugar product may be used as such or may befurther processed for example purified.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Cellulose is a linear polysaccharide composed of glucose residues linkedby β-1,4 bonds. The linear nature of the cellulose fibers, as well asthe stoichiometry of the β-linked glucose (relative to α) generatesstructures more prone to inter strand hydrogen bonding than the highlybranched α-linked structures of starch. Thus, cellulose polymers aregenerally less soluble, and form more tightly bound fibers than thefibers found in starch.

Enzymes that may be included in the stable enzyme composition used inthe invention are now described in more detail:

Lytic polysaccharide monooxygenases such as GH61, endoglucanases (EG)and exo-cellobiohydrolases (CBH) catalyze the hydrolysis of insolublecellulose to products such as cellooligosaccharides (cellobiose as amain product), while beta-glucosidases (BG) convert theoligosaccharides, mainly cellobiose and cellotriose, to glucose.

Hemicellulose is a complex polymer, and its composition often varieswidely from organism to organism and from one tissue type to another. Ingeneral, a main component of hemicellulose is β-1,4-linked xylose, afive carbon sugar. However, this xylose is often branched at 0 to 3and/or 0 to 2 atom of xylose, and can be substituted with linkages toarabinose, galactose, mannose, glucuronic acid, galacturonic acid or byesterification to acetic acid (and esterification of ferulic acid toarabinose). Hemicellulose can also contain glucan, which is a generalterm for β-linked six carbon sugars (such as the β-(1,3)(1,4) glucansand heteroglucans mentioned previously) and additionally glucomannans(in which both glucose and mannose are present in the linear backbone,linked to each other by β-linkages).

Xylanases together with other accessory enzymes, for exampleα-L-arabinofuranosidases, feruloyl and acetylxylan esterases,glucuronidases, and β-xylosidases catalyze the hydrolysis ofhemicelluloses.

Pectic substances include pectins, arabinans, galactans andarabinogalactans. Pectins are the most complex polysaccharides in theplant cell wall. They are built up around a core chain of α(1,4)-linkedD-galacturonic acid units interspersed to some degree with L-rhamnose.In any one cell wall there are a number of structural units that fitthis description and it has generally been considered that in a singlepectic molecule, the core chains of different structural units arecontinuous with one another.

The principal types of structural unit are: galacturonan(homogalacturonan), which may be substituted with methanol on thecarboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I (RGI),in which galacturonic acid units alternate with rhamnose units carrying(1,4)-linked galactan and (1,5)-linked arabinan side-chains. Thearabinan side-chains may be attached directly to rhamnose or indirectlythrough the galactan chains; xylogalacturonan, with single xylosyl unitson O-3 of galacturonic acid (closely associated with RGI); andrhamnogalacturonan II (RGII), a particularly complex minor unitcontaining unusual sugars, for example apiose. An RGII unit may containtwo apiosyl residues which, under suitable ionic conditions, canreversibly form esters with borate.

A composition for use in a method of the invention comprises preferablyat least two activities, although typically a composition will comprisemore than two activities, for example, three, four, five, six, seven,eight, nine or more. Typically, a composition of the invention maycomprise at least two different cellulases or one cellulase and at leastone hemicellulase. A composition of the invention may comprisecellulases, but no xylanases. In addition, a composition of theinvention may comprise auxiliary enzyme activity, i.e. additionalactivity which, either directly or indirectly, leads to lignocellulosedegradation. Examples of such auxiliary activities are mentioned herein.

Thus, a composition for use in the invention may comprise GH61,endoglucanase activity and/or cellobiohydrolase activity and/orbeta-glucosidase activity. A composition for use in the invention maycomprise more than one enzyme activity in one or more of those classes.For example, a composition for use in the invention may comprise twoendoglucanase activities, for example, endo-1,3(1,4)-β glucanaseactivity and endo-β-1,4-glucanase activity. Such a composition may alsocomprise one or more xylanase activities. Such a composition maycomprise an auxiliary enzyme activity.

A composition for use in the current invention may be derived from afungus, such as a filamentous fungus such as Rasamsonia, such asRasamsonia emersonii. In an embodiment a core set of (lignocellulosedegrading) enzyme activities may be derived from Rasamsonia emersonii.Rasamsonia emersonii can provide a highly effective set of activities asdemonstrated herein for the hydrolysis of lignocellulosic material. Ifneeded, the set of activities can be supplemented with additional enzymeactivities from other sources. Such additional activities may be derivedfrom classical sources and/or produced by a genetically modifiedorganisms.

The activities in a composition for use in the invention may bethermostable. Herein, this means that the activity has a temperatureoptimum of about 60° C. or higher, for example about 70° C. or higher,such as about 75° C. or higher, for example about 80° C. or higher suchas 85° C. or higher. Activities in a composition for use in theinvention will typically not have the same temperature optima, butpreferably will, nevertheless, be thermostable.

In addition, enzyme activities in a composition for use in the inventionmay be able to work at low pH. For the purposes of this invention, lowpH indicates a pH of about 5.5 or lower, about 5 or lower, about 4.9 orlower, about 4.8 or lower, about 4.7 or lower, about 4,6 or lower, about4.5 or lower, about 4.4 or lower, about 4.3 or lower, about 4.2 orlower, about 4,1 or lower, about 4.0 or lower, about 3.9 or lower, orabout 3.8 or lower, about 3.7 or lower, about 3.6 or lower, or about 3.5or lower.

Activities in a composition for use in the invention may be defined by acombination of any of the above temperature optima and pH values.

The composition used in a method of the invention may comprise, inaddition to the activities derived from Rasamsonia, a cellulase (forexample one derived from a source other than Rasamsonia) and/or ahemicellulase (for example one derived from a source other thanRasamsonia) and/or a pectinase.

The enzyme composition for use in the current invention may comprise acellulase and/or a hemicellulase and/or a pectinase from a source otherthan Rasamsonia.

For example, a composition for use in the invention may comprise abeta-glucosidase (BG) from Aspergillus, such as Aspergillus oryzae, suchas the one disclosed in WO 02/095014 or the fusion protein havingbeta-glucosidase activity disclosed in WO 2008/057637, or Aspergillusfumigatus, such as the one disclosed as SEQ ID NO:2 in WO 2005/047499 orSEQ ID NO:5 in WO 2014/130812 or an Aspergillus fumigatusbeta-glucosidase variant, such as one disclosed in WO 2012/044915, suchas one with the following substitutions: F100D, S283G, N456E, F512Y(using SEQ ID NO: 5 in WO 2014/130812 for numbering), or Aspergillusaculeatus, Aspergillus niger or Aspergillus kawachi. In anotherembodiment the beta-glucosidase is derived from Penicillium, such asPenicillium brasilianum disclosed as SEQ ID NO:2 in WO 2007/019442, orfrom Trichoderma, such as Trichoderma reesei, such as ones described inU.S. Pat. Nos. 6,022,725, 6,982,159, 7,045,332, 7,005,289, US2006/0258554 US 2004/0102619. In an embodiment even a bacterialbeta-glucosidase can be used. In another embodiment the beta-glucosidaseis derived from Thielavia terrestris (WO 2011/035029) or Trichophaeasaccata (WO 2007/019442).

For example, a composition for use in the invention may comprise anendoglucanase (EG) from Trichoderma, such as Trichoderma reesei; fromHumicola, such as a strain of Humicola insolens; from Aspergillus, suchas Aspergillus aculeatus or Aspergillus kawachii; from Erwinia, such asErwinia carotovara; from Fusarium, such as Fusarium oxysporum; fromThielavia, such as Thielavia terrestris; from Humicola, such as Humicolagrisea var. thermoidea or Humicola insolens; from Melanocarpus, such asMelanocarpus albomyces; from Neurospora, such as Neurospora crassa; fromMyceliophthora, such as Myceliophthora thermophila; from Cladorrhinum,such as Cladorrhinum foecundissimum and/or from Chrysosporium, such as astrain of Chrysosporium lucknowense. In an embodiment even a bacterialendoglucanase can be used including, but are not limited to,Acidothermus cellulolyticus endoglucanase (see WO 91/05039; WO 93/15186;U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (see WO05/093050); and Thermobifida fusca endoglucanase V (see WO 05/093050).

For example, a composition for use in the invention may comprise acellobiohydrolase I from Aspergillus, such as Aspergillus fumigatus,such as the Cel7A CBH I disclosed in SEQ ID NO:6 in WO 2011/057140 orSEQ ID NO:6 in WO 2014/130812, or from Trichoderma, such as Trichodermareesei.

For example, a composition for use in the invention may comprise acellobiohydrolase II from Aspergillus, such as Aspergillus fumigatus,such as the one in SEQ ID NO:7 in WO 2014/130812 or from Trichoderma,such as Trichoderma reesei, or from Thielavia, such as Thielaviaterrestris, such as cellobiohydrolase II CEL6A from Thielaviaterrestris.

For example, a composition for use in the invention may comprise a GH61polypeptide (a lytic polysaccharide monooxygenase) from Thermoascus,such as Thermoascus aurantiacus, such as the one described in WO2005/074656 as SEQ ID NO:2 and SEQ ID NO:1 in WO2014/130812 and in WO2010/065830; or from Thielavia, such as Thielavia terrestris, such asthe one described in WO 2005/074647 as SEQ ID NO: 8 or SEQ ID NO:4 inWO2014/130812 and in WO 2008/148131, and WO 2011/035027; or fromAspergillus, such as Aspergillus fumigatus, such as the one described inWO 2010/138754 as SEQ ID NO:2 or SEQ ID NO: 3 in WO2014/130812; or fromPenicillium, such as Penicillium emersonii, such as the one disclosed asSEQ ID NO:2 in WO 2011/041397 or SEQ ID NO:2 in WO2014/130812. Othersuitable GH61 polypeptides include, but are not limited to, Trichodermareesei (see WO 2007/089290), Myceliophthora thermophila (see WO2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868),Penicillium pinophilum (see WO 2011/005867), Thermoascus sp. (see WO2011/039319), and Thermoascus crustaceous (see WO 2011/041504). In oneaspect, the GH61 polypeptide is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043, e.g.manganese sulfate. In one aspect, the GH61 polypeptide is used in thepresence of a dioxy compound, a bicylic compound, a heterocycliccompound, a nitrogen-containing compound, a quinone compound, asulfur-containing compound, or a liquor obtained from a pretreatedcellulosic material such as pretreated corn stover.

Other cellulolytic enzymes that may be used in a composition for use inthe invention are described in WO 98/13465, WO 98/015619, WO 98/015633,WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO2003/052057, WO 2003/052118, WO 2004/016760, WO 2004/043980, WO2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO2005/093073, WO 2006/074005, WO 2006/117432, WO 2007/071818, WO2007/071820, WO 2008/008070, WO 2008/008793, U.S. Pat. Nos. 5,457,046,5,648,263, and 5,686,593, to name just a few.

In addition, examples of xylanases useful in the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(see WO 94/21785), Aspergillus fumigatus (see WO 2006/078256),Penicillium pinophilum (see WO 2011/041405), Penicillium sp. (see WO2010/126772), Thielavia terrestris NRRL 8126 (see WO 2009/079210), andTrichophaea saccata GH10 (see WO 2011/057083). Examples ofbeta-xylosidases useful in the integrated processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa and Trichoderma reesei. Examples of acetylxylanesterases useful in the present invention include, but are not limitedto, acetylxylan esterases from Aspergillus aculeatus (see WO2010/108918), Chaetomium globosum, Chaetomium gracile, Humicola insolensDSM 1800 (see WO 2009/073709), Hypocrea jecorina (see WO 2005/001036),Myceliophtera thermophila (see WO 2010/014880), Neurospora crassa,Phaeosphaeria nodorum and Thielavia terrestris NRRL 8126 (see WO2009/042846). Examples of feruloyl esterases (ferulic acid esterases)useful in the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (see WO 2009/076122),Neosartorya fischeri, Neurospora crassa, Penicillium aurantiogriseum(see WO 2009/127729), and Thielavia terrestris (see WO 2010/053838 andWO 2010/065448). Examples of arabinofuranosidases useful in the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger, Humicola insolens DSM 1800 (see WO 2006/114094 and WO2009/073383) and M. giganteus (see WO 2006/114094). Examples ofalpha-glucuronidases useful in the present invention include, but arenot limited to, alpha-glucuronidases from Aspergillus clavatus,Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Humicolainsolens (see WO 2010/014706), Penicillium aurantiogriseum (see WO2009/068565) and Trichoderma reesei.

A composition for use in the invention may comprise one, two, three,four classes or more of cellulase, for example one, two three or four orall of a GH61, an endoglucanase (EG), one or two exo-cellobiohydrolase(CBH) and a beta-glucosidase (BG). A composition for use in theinvention may comprise two or more of any of these classes of cellulase.

A composition of the invention may comprise an activity which has adifferent type of cellulase activity and/or hemicellulase activityand/or pectinase activity than that provided by the composition for usein a method of the invention. For example, a composition of theinvention may comprise one type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by a composition asdescribed herein and a second type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by an additionalcellulase/hemicellulase/pectinase.

Herein, a cellulase is any polypeptide which is capable of degrading ormodifying cellulose. A polypeptide which is capable of degradingcellulose is one which is capable of catalyzing the process of breakingdown cellulose into smaller units, either partially, for example intocellodextrins, or completely into glucose monomers. A cellulaseaccording to the invention may give rise to a mixed population ofcellodextrins and glucose monomers when contacted with the cellulase.Such degradation will typically take place by way of a hydrolysisreaction.

Lytic polysaccharide monooxygenases (LPMO) are recently classified byCAZy in family AA9 (Auxiliary Activity Family 9) or family AA10(Auxiliary Activity Family 10). As mentioned above, lytic polysaccharidemonooxygenases are able to open a crystalline glucan structure. Lyticpolysaccharide monooxygenases may also affect cello-oligosaccharides.PMO and LPMO are used herein interchangeably. GH61 (glycoside hydrolasefamily 61 or sometimes referred to EGIV) proteins are oxygen-dependentpolysaccharide monooxygenases (PMO's) according to the latestliterature. Often in literature, these proteins are mentioned to enhancethe action of cellulases on lignocellulose substrates. GH61 wasoriginally classified as endoglucanase based on measurement of very weakendo-1,413-d-glucanase activity in one family member. The term “GH61” asused herein, is to be understood as a family of enzymes, which sharecommon conserved sequence portions and foldings to be classified infamily 61 of the well-established CAZY GH classification system(http://www.cazy.org/GH61.html). The glycoside hydrolase family 61 is amember of the family of glycoside hydrolases EC 3.2.1. GH61 is usedherein as being part of the cellulases. CBM33 (family 33carbohydrate-binding module) is a lytic polysaccharide monooxygenase(see lsaksen et al, Journal of Biological Chemistry, vol. 289, no. 5,pp. 2632-2642), CAZy has recently reclassified CBM33 in AA10 (AuxiliaryActivity Family 10).

Herein, a hemicellulase is any polypeptide which is capable of degradingor modifying hemicellulose. That is to say, a hemicellulase may becapable of degrading or modifying one or more of xylan, glucuronoxylan,arabinoxylan, glucomannan and xyloglucan. A polypeptide which is capableof degrading a hemicellulose is one which is capable of catalyzing theprocess of breaking down the hemicellulose into smaller polysaccharides,either partially, for example into oligosaccharides, or completely intosugar monomers, for example hexose or pentose sugar monomers. Ahemicellulase according to the invention may give rise to a mixedpopulation of oligosaccharides and sugar monomers when contacted withthe hemicellulase. Such degradation will typically take place by way ofa hydrolysis reaction.

Herein, a pectinase is any polypeptide which is capable of degrading ormodifying pectin. A polypeptide which is capable of degrading pectin isone which is capable of catalyzing the process of breaking down pectininto smaller units, either partially, for example into oligosaccharides,or completely into sugar monomers. A pectinase according to theinvention may give rise to a mixed population of oligosacchardies andsugar monomers when contacted with the pectinase. Such degradation willtypically take place by way of a hydrolysis reaction.

Accordingly, a composition of the invention may comprise any cellulase,for example, a GH61, a cellobiohydrolase, an endo-β-1,4-glucanase, abeta-glucosidase or a β-(1,3)(1,4)-glucanase.

Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which iscapable of catalysing the hydrolysis of 1,4-β-D-glucosidic linkages incellulose or cellotetraose, releasing cellobiose from the ends of thechains. This enzyme may also be referred to as cellulose,1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucancellobiohydrolase, avicelase, exo-1,4-β-D-glucanase,exocellobiohydrolase or exoglucanase.

Herein, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-β-D-glucosidic linkagesin cellulose, lichenin or cereal β-D-glucans. Such a polypeptide mayalso be capable of hydrolyzing 1,4-linkages in β-D-glucans alsocontaining 1,3-linkages. This enzyme may also be referred to ascellulase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase,carboxymethyl cellulase, celludextrinase, endo-1,4-β-D-glucanase,endo-1,4-β-D-glucanohydrolase, endo-1,4-β-glucanase or endoglucanase.

Herein, a beta-glucosidase (EC 3.2.1.21) is any polypeptide which iscapable of catalysing the hydrolysis of terminal, non-reducingβ-D-glucose residues with release of β-D-glucose. Such a polypeptide mayhave a wide specificity for β-D-glucosides and may also hydrolyze one ormore of the following: a β-D-galactoside, an α-L-arabinoside, aβ-D-xyloside or a β-D-fucoside. This enzyme may also be referred to asamygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

Herein a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide whichis capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic linkagesin β-D-glucans containing 1,3- and 1,4-bonds. Such a polypeptide may acton lichenin and cereal β-D-glucans, but not on β-D-glucans containingonly 1,3- or 1,4-bonds. This enzyme may also be referred to aslicheninase, 1,3-1,4-β-D-glucan 4-glucanohydrolase, β-glucanase,endo-β-1,3-1,4 glucanase, lichenase or mixed linkage β-glucanase. Analternative for this type of enzyme is EC 3.2.1.6, which is described asendo-1,3(4)-beta-glucanase. This type of enzyme hydrolyses 1,3- or1,4-linkages in beta-D-glucanse when the glucose residue whose reducinggroup is involved in the linkage to be hydrolysed is itself substitutedat C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase,1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates includelaminarin, lichenin and cereal beta-D-glucans.

A composition of the invention may comprise any hemicellulase, forexample, an endoxylanase, a β-xylosidase, a α-L-arabionofuranosidase, anα-D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, acoumaroyl esterase, an α-galactosidase, a β-galactosidase, a β-mannanaseor a β-mannosidase.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endohydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyse1,4 xylosidic linkages in glucuronoarabinoxylans.

Herein, a β-xylosidase (EC 3.2.1.37) is any polypeptide which is capableof catalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase orxylobiase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptide which iscapable of catalyzing a reaction of the following form:alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzyme mayalso be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyze 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. Alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyzes thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

Herein, an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide whichis capable of catalyzing the deacetylation of xylans andxylo-oligosaccharides. Such a polypeptide may catalyze the hydrolysis ofacetyl groups from polymeric xylan, acetylated xylose, acetylatedglucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically,not from triacetylglycerol. Such a polypeptide typically does not act onacetylated mannan or pectin.

Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:feruloyl-saccharide+H(2)O=ferulate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. It may typicallycatalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

Herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. This enzyme mayalso be referred to as trans-4-coumaroyl esterase, trans-p-coumaroylesterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzymealso falls within EC 3.1.1.73 so may also be referred to as a feruloylesterase.

Herein, an α-galactosidase (EC 3.2.1.22) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

Herein, a β-galactosidase (EC 3.2.1.23) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingβ-D-galactose residues in β-D-galactosides. Such a polypeptide may alsobe capable of hydrolyzing α-L-arabinosides. This enzyme may also bereferred to as exo-(1->4)-β-D-galactanase or lactase. Herein, aβ-mannanase (EC 3.2.1.78) is any polypeptide which is capable ofcatalyzing the random hydrolysis of 1,4-β-D-mannosidic linkages inmannans, galactomannans and glucomannans. This enzyme may also bereferred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.

Herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

A composition of the invention may comprise any pectinase, for examplean endo polygalacturonase, a pectin methyl esterase, anendo-galactanase, a beta galactosidase, a pectin acetyl esterase, anendo-pectin lyase, pectate lyase, alpha rhamnosidase, anexo-galacturonase, an expolygalacturonate lyase, a rhamnogalacturonanhydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetylesterase, a rhamnogalacturonan galacturonohydrolase, axylogalacturonase.

Herein, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide whichis capable of catalyzing the random hydrolysis of1,4-α-D-galactosiduronic linkages in pectate and other galacturonans.This enzyme may also be referred to as polygalacturonase pectindepolymerase, pectinase, endopolygalacturonase, pectolase, pectinhydrolase, pectin polygalacturonase, poly-α-1,4-galacturonideglycanohydrolase, endogalacturonase; endo-D-galacturonase orpoly(1,4-α-D-galacturonide) glycanohydrolase.

Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme which iscapable of catalyzing the reaction: pectin+n H₂O=n methanol+pectate. Theenzyme may also been known as pectinesterase, pectin demethoxylase,pectin methoxylase, pectin methylesterase, pectase, pectinoesterase orpectin pectylhydrolase.

Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capable ofcatalyzing the endohydrolysis of 1,4-β-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

Herein, a pectin acetyl esterase is defined herein as any enzyme whichhas an acetyl esterase activity which catalyzes the deacetylation of theacetyl groups at the hydroxyl groups of GaIUA residues of pectin

Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable ofcatalyzing the eliminative cleavage of (1→4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-β-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducingends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-β-O-methyl-α-D-galacturonan lyase.

Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzingthe eliminative cleavage of (1→4)-α-D-galacturonan to giveoligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-α-D-galacturonan lyase.

Herein, an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingα-L-rhamnose residues in α-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T,α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.

Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable ofhydrolysis of pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known asexo-poly-α-galacturonosidase, exopolygalacturonosidase orexopolygalacturanosidase.

Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable ofcatalyzing:(1,4-α-D-galacturonide)_(n)+H₂O=(1,4-α-D-galacturonide)_(n+1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-α-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase orpoly(1,4-α-D-galacturonide) galacturonohydrolase.

Herein, exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptidecapable of catalyzing eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate, i.e. de-esterified pectin. This enzyme may be known aspectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exopectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonanreducing-end-disaccharide-lyase.

Herein, rhamnogalacturonan hydrolase is any polypeptide which is capableof hydrolyzing the linkage between galactosyluronic acid acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

Herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleaving α-L-Rhap-(1→4)-α-D-GalpAlinkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

Herein, rhamnogalacturonan acetyl esterase is any polypeptide whichcatalyzes the deacetylation of the backbone of alternating rhamnose andgalacturonic acid residues in rhamnogalacturonan.

Herein, rhamnogalacturonan galacturonohydrolase is any polypeptide whichis capable of hydrolyzing galacturonic acid from the non-reducing end ofstrictly alternating rhamnogalacturonan structures in an exo-fashion.

Herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the β-xylose substituted galacturonic acidbackbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide which iscapable of catalyzing endohydrolysis of 1,5-α-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be known asendo-arabinase, arabinan endo-1,5-α-L-arabinosidase,endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.

A composition of the invention will typically comprise at least onecellulase and/or at least one hemicellulase and/or at least onepectinase (one of which is a polypeptide according to the invention). Acomposition of the invention may comprise a GH61, a cellobiohydrolase,an endoglucanase and/or a beta-glucosidase. Such a composition may alsocomprise one or more hemicellulases and/or one or more pectinases.

In addition, one or more (for example two, three, four or all) of anamylase, a protease, a lipase, a ligninase, a hexosyltransferase, aglucuronidase or an expansin or a cellulose induced protein or acellulose integrating protein or like protein may be present in acomposition of the invention (these are referred to as auxiliaryactivities above).

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the inventionincorporated herein by reference. Some specific types of proteasesinclude, cysteine proteases including pepsin, papain and serineproteases including chymotrypsins, carboxypeptidases andmetalloendopeptidases.

“Lipase” includes enzymes that hydrolyze lipids, fatty acids, andacylglycerides, including phospoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

“Ligninase” includes enzymes that can hydrolyze or break down thestructure of lignin polymers. Enzymes that can break down lignin includelignin peroxidases, manganese peroxidases, laccases and feruloylesterases, and other enzymes described in the art known to depolymerizeor otherwise break lignin polymers. Also included are enzymes capable ofhydrolyzing bonds formed between hemicellulosic sugars (notablyarabinose) and lignin. Ligninases include but are not limited to thefollowing group of enzymes: lignin peroxidases (EC 1.11.1.14), manganeseperoxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloylesterases (EC 3.1.1.73).

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable ofcatalyzing a transferase reaction, but which can also catalyze ahydrolysis reaction, for example of cellulose and/or cellulosedegradation products. An example of a hexosyltransferase which may beused in the invention is a β-glucanosyltransferase. Such an enzyme maybe able to catalyze degradation of (1,3)(1,4)glucan and/or celluloseand/or a cellulose degradation product.

“Glucuronidase” includes enzymes that catalyze the hydrolysis of aglucoronoside, for example β-glucuronoside to yield an alcohol. Manyglucuronidases have been characterized and may be suitable for use inthe invention, for example β-glucuronidase (EC 3.2.1.31),hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamineglucuronidase (3.2.1.56), glycyrrhizinate β-glucuronidase (3.2.1.128) orα-D-glucuronidase (EC 3.2.1.139).

A composition for use in the invention may comprise an expansin orexpansin-like protein, such as a swollenin (see Salheimo et al., Eur. J.Biohem. 269, 4202-4211, 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this invention, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars.

A composition for use in the invention may be a cellulose inducedprotein, for example the polypeptide product of the cip1 or cip2 gene orsimilar genes (see Foreman et al., J. Biol. Chem. 278(34), 31988-31997,2003), a cellulose/cellulosome integrating protein, for example thepolypeptide product of the cipA or cipC gene, or a scaffoldin or ascaffoldin-like protein. Scaffoldins and cellulose integrating proteinsare multi-functional integrating subunits which may organizecellulolytic subunits into a multi-enzyme complex. This is accomplishedby the interaction of two complementary classes of domain, i.e. acohesion domain on scaffoldin and a dockerin domain on each enzymaticunit. The scaffoldin subunit also bears a cellulose-binding module (CBM)that mediates attachment of the cellulosome to its substrate. Ascaffoldin or cellulose integrating protein for the purposes of thisinvention may comprise one or both of such domains.

A composition for use in the current invention may also comprise acatalase. The term “catalase” means a hydrogen-peroxide:hydrogen-peroxide oxidoreductase (EC 1.11.1.6 or EC 1.11.1.21) thatcatalyzes the conversion of two hydrogen peroxides to oxygen and twowaters. Catalase activity can be determined by monitoring thedegradation of hydrogen peroxide at 240 nm based on the followingreaction: 2H₂O₂→2H₂O+O₂. The reaction is conducted in 50 mM phosphate pH7.0 at 25° C. with 10.3 mM substrate (H₂O₂) and approximately 100 unitsof enzyme per ml. Absorbance is monitored spectrophotometrically within16-24 seconds, which should correspond to an absorbance reduction from0.45 to 0.4. One catalase activity unit can be expressed as onemicromole of H₂O₂ degraded per minute at pH 7.0 and 25° C.

A composition for use in a method of the invention may be composed of amember of each of the classes of enzymes mentioned above, severalmembers of one enzyme class, or any combination of these enzymes classesor helper proteins (i.e. those proteins mentioned herein which do nothave enzymatic activity per se, but do nevertheless assist inlignocellulosic degradation).

A composition for use in a method of the invention may be composed ofenzymes from (1) commercial suppliers; (2) cloned genes expressingenzymes; (3) complex broth (such as that resulting from growth of amicrobial strain in media, wherein the strains secrete proteins andenzymes into the media; (4) cell lysates of strains grown as in (3);and/or (5) plant material expressing enzymes. Different enzymes in acomposition of the invention may be obtained from different sources.

The enzymes can be produced either exogenously in microorganisms,yeasts, fungi, bacteria or plants, then isolated and added, for example,to lignocellulosic feedstock. Alternatively, the enzymes are produced,but not isolated, and crude cell mass fermentation broth, or plantmaterial (such as corn stover or wheat straw), and the like may be addedto, for example, the feedstock. Alternatively, the crude cell mass orenzyme production medium or plant material may be treated to preventfurther microbial growth (for example, by heating or addition ofantimicrobial agents), then added to, for example, a feedstock. Thesecrude enzyme mixtures may include the organism producing the enzyme.Alternatively, the enzyme may be produced in a fermentation that uses(pre-treated) feedstock (such as corn stover or wheat straw) to providenutrition to an organism that produces an enzyme(s). In this manner,plants that produce the enzymes may themselves serve as alignocellulosic feedstock and be added into lignocellulosic feedstock.

In the uses and methods described herein, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

The invention thus relates to methods in which the composition describedabove are used and to uses of the composition in industrial processes.

In an embodiment the enzyme compositions may be a whole fermentationbroth as described below. The whole fermentation broth can be preparedfrom fermentation of non-recombinant and/or recombinant filamentousfungi. In an embodiment the filamentous fungus is a recombinantfilamentous fungus comprising one or more genes which can be homologousor heterologous to the filamentous fungus. In an embodiment, thefilamentous fungus is a recombinant filamentous fungus comprising one ormore genes which can be homologous or heterologous to the filamentousfungus wherein the one or more genes encode enzymes that can degrade acellulosic substrate. The whole fermentation broth may comprise any ofthe polypeptides or any combination thereof.

Preferably, the enzyme composition is whole fermentation broth whereinthe cells are killed. The whole fermentation broth may contain organicacid(s) (used for killing the cells), killed cells and/or cell debris,and culture medium.

Generally, the filamentous fungi is cultivated in a cell culture mediumsuitable for production of enzymes capable of hydrolyzing a cellulosicsubstrate. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable culture media, temperature rangesand other conditions suitable for growth and cellulase and/orhemicellulase and/or pectinase production are known in the art. Thewhole fermentation broth can be prepared by growing the filamentousfungi to stationary phase and maintaining the filamentous fungi underlimiting carbon conditions for a period of time sufficient to expressthe one or more cellulases and/or hemicellulases and/or pectinases. Onceenzymes, such as cellulases and/or hemicellulases and/or pectinases, aresecreted by the filamentous fungi into the fermentation medium, thewhole fermentation broth can be used. The whole fermentation broth ofthe present invention may comprise filamentous fungi. In someembodiments, the whole fermentation broth comprises the unfractionatedcontents of the fermentation materials derived at the end of thefermentation. Typically, the whole fermentation broth comprises thespent culture medium and cell debris present after the filamentous fungiis grown to saturation, incubated under carbon-limiting conditions toallow protein synthesis (particularly, expression of cellulases and/orhemicellulases and/or pectinases). In some embodiments, the wholefermentation broth comprises the spent cell culture medium,extracellular enzymes and filamentous fungi. In some embodiments, thefilamentous fungi present in whole fermentation broth can be lysed,permeabilized, or killed using methods known in the art to produce acell-killed whole fermentation broth. In an embodiment, the wholefermentation broth is a cell-killed whole fermentation broth, whereinthe whole fermentation broth containing the filamentous fungi cells arelysed or killed. In some embodiments, the cells are killed by lysing thefilamentous fungi by chemical and/or pH treatment to generate thecell-killed whole broth of a fermentation of the filamentous fungi. Insome embodiments, the cells are killed by lysing the filamentous fungiby chemical and/or pH treatment and adjusting the pH of the cell-killedfermentation mix to a suitable pH. In an embodiment, the wholefermentation broth comprises a first organic acid component comprisingat least one 1-5 carbon organic acid and/or a salt thereof and a secondorganic acid component comprising at least 6 or more carbon organic acidand/or a salt thereof. In an embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or any combination thereof and the second organic acid component isbenzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid,phenylacetic acid, a salt thereof, or any combination thereof.

The term “whole fermentation broth” as used herein refers to apreparation produced by cellular fermentation that undergoes no orminimal recovery and/or purification. For example, whole fermentationbroths are produced when microbial cultures are grown to saturation,incubated under carbon-limiting conditions to allow protein synthesis(e.g., expression of enzymes by host cells) and secretion into cellculture medium. Typically, the whole fermentation broth isunfractionated and comprises spent cell culture medium, extracellularenzymes, and microbial, preferably non-viable, cells.

If needed, the whole fermentation broth can be fractionated and the oneor more of the fractionated contents can be used. For instance, thekilled cells and/or cell debris can be removed from a whole fermentationbroth to provide a composition that is free of these components.

The whole fermentation broth may further comprise a preservative and/oranti-microbial agent. Such preservatives and/or agents are known in theart.

The whole fermentation broth as described herein is typically a liquid,but may contain insoluble components, such as killed cells, cell debris,culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedwhole fermentation broth.

In an embodiment, the whole fermentation broth may be supplemented withone or more enzyme activities that are not expressed endogenously, orexpressed at relatively low level by the filamentous fungi, to improvethe degradation of the cellulosic substrate, for example, to fermentablesugars such as glucose or xylose. The supplemental enzyme(s) can beadded as a supplement to the whole fermentation broth and the enzymesmay be a component of a separate whole fermentation broth, or may bepurified, or minimally recovered and/or purified.

In an embodiment, the whole fermentation broth comprises a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. Alternatively, the whole fermentation broth cancomprise a mixture of a whole fermentation broth of a fermentation of anon-recombinant filamentous fungus and a recombinant filamentous fungusoverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. In an embodiment, the whole fermentation brothcomprises a whole fermentation broth of a fermentation of a filamentousfungi overexpressing beta-glucosidase. Alternatively, the wholefermentation broth for use in the present methods and reactivecompositions can comprise a mixture of a whole fermentation broth of afermentation of a non-recombinant filamentous fungus and a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing a beta-glucosidase.

Lignocellulosic Material

Lignocellulosic material herein includes any lignocellulosic and/orhemicellulosic material. Lignocellulosic material suitable for use asfeedstock in the invention includes biomass, e.g. virgin biomass and/ornon-virgin biomass such as agricultural biomass, commercial organics,construction and demolition debris, municipal solid waste, waste paperand yard waste. Common forms of biomass include trees, shrubs andgrasses, wheat, wheat straw, sugar cane, cane straw, sugar cane bagasse,switch grass, miscanthus, energy cane, corn, corn stover, corn husks,corn cobs, canola stems, soybean stems, sweet sorghum, corn kernelincluding fiber from kernels, products and by-products from milling ofgrains such as corn, wheat and barley (including wet milling and drymilling) often called “bran or fibre” as well as municipal solid waste,waste paper and yard waste. The biomass can also be, but is not limitedto, herbaceous material, agricultural residues, forestry residues,municipal solid wastes, waste paper, and pulp and paper mill residues.“Agricultural biomass” includes branches, bushes, canes, corn and cornhusks, energy crops, forests, fruits, flowers, grains, grasses,herbaceous crops, leaves, bark, needles, logs, roots, saplings, shortrotation woody crops, shrubs, switch grasses, trees, vegetables, fruitpeels, vines, sugar beet pulp, wheat midlings, oat hulls, and hard andsoft woods (not including woods with deleterious materials). Inaddition, agricultural biomass includes organic waste materialsgenerated from agricultural processes including farming and forestryactivities, specifically including forestry wood waste. Agriculturalbiomass may be any of the aforementioned singularly or in anycombination or mixture thereof.

Pretreatment

The lignocellulosic material used in the present invention may be washedand/or pretreated. The feedstock may optionally be pretreated with heat,mechanical and/or chemical modification or any combination of suchmethods in order to enhance the accessibility of the substrate toenzymatic hydrolysis and/or hydrolyse the hemicellulose and/orsolubilize the hemicellulose and/or cellulose and/or lignin, in any wayknown in the art. In one embodiment, the pr-treatment is conductedtreating the lignocellulose with steam explosion, hot water treatment ortreatment with dilute acid or dilute base.

In an embodiment the lignocellulosic material is pretreated beforeand/or during the enzymatic hydrolysis. Pretreatment methods are knownin the art and include, but are not limited to, heat, mechanical,chemical modification, biological modification and any combinationthereof. Pretreatment is typically performed in order to enhance theaccessibility of the lignocellulosic material to enzymatic hydrolysisand/or hydrolyse the hemicellulose and/or solubilize the hemicelluloseand/or cellulose and/or lignin, in the lignocellulosic material. In anembodiment, the pretreatment comprises treating the lignocellulosicmaterial with steam explosion, hot water treatment or treatment withdilute acid or dilute base. Examples of pretreatment methods include,but are not limited to, steam treatment (e.g. treatment at 100-260° C.,at a pressure of 7-45 bar, at neutral pH, for 1-10 minutes), dilute acidtreatment (e.g. treatment with 0.1-5% H₂SO₄ and/or SO₂ and/or HNO₃and/or HCl, in presence or absence of steam, at 120-200° C., at apressure of 2-15 bar, at acidic pH, for 2-30 minutes), organosolvtreatment (e.g. treatment with 1-1.5% H₂SO₄ in presence of organicsolvent and steam, at 160-200° C., at a pressure of 7-30 bar, at acidicpH, for 30-60 minutes), lime treatment (e.g. treatment with 0.1-2%NaOH/Ca(OH)₂ in the presence of water/steam at 60-160° C., at a pressureof 1-10 bar, at alkaline pH, for 60-4800 minutes), ARP treatment (e.g.treatment with 5-15% NH₃, at 150-180° C., at a pressure of 9-17 bar, atalkaline pH, for 10-90 minutes), AFEX treatment (e.g. treatmentwith >15% NH₃, at 60-140° C., at a pressure of 8-20 bar, at alkaline pH,for 5-30 minutes).

Washing Step

Optionally, the process according to the invention comprises a washingstep. The optional washing step may be used to remove water solublecompounds that may act as inhibitors for the fermentation step. Thewashing step may be conducted in known manner.

The lignocellulosic material may be washed. In an embodiment thelignocellulosic material may be washed after the pretreatment. Thewashing step may be used to remove water soluble compounds that may actas inhibitors for the fermentation and/or hydrolysis step. The washingstep may be conducted in manner known to the skilled person. Next towashing, other detoxification methods do exist. The pretreatedlignocellulosic material may also be detoxified by any (or anycombination) of these methods which include, but are not limited to,solid/liquid separation, vacuum evaporation, extraction, adsorption,neutralization, overliming, addition of reducing agents, addition ofdetoxifying enzymes such as laccases or peroxidases, addition ofmicroorganisms capable of detoxification of hydrolysates.

Enzymatic Hydrolysis

The enzyme composition used in the process of the invention canextremely effectively hydrolyze lignocellulolytic material, for examplecorn stover, wheat straw, cane straw, and/or sugar cane bagasse, whichcan then be further converted into a useful product, such as ethanol,biogas, butanol, lactic acid, a plastic, an organic acid, a solvent, ananimal feed supplement, a pharmaceutical, a vitamin, an amino acid, anenzyme or a chemical feedstock. Additionally, intermediate products froma process following the hydrolysis, for example lactic acid asintermediate in biogas production, can be used as building block forother materials. The present invention is exemplified with theproduction of ethanol but this is done as exemplification only ratherthan as limitation, the other mentioned useful products can be producedequally well.

The process according to the invention comprises an enzymatic hydrolysisstep. The enzymatic hydrolysis includes, but is not limited to,hydrolysis for the purpose of liquefaction of the feedstock andhydrolysis for the purpose of releasing sugar from the feedstock orboth. In this step optionally pretreated and optionally washedlignocellulosic material is brought into contact with the enzymecomposition according to the invention. Depending on the lignocellulosicmaterial and the pretreatment, the different reaction conditions, e.g.temperature, enzyme dosage, hydrolysis reaction time and dry matterconcentration, may be adapted by the skilled person in order to achievea desired conversion of lignocellulose to sugar. Some indications aregiven hereafter.

In one aspect of the invention the hydrolysis is conducted at atemperature of 45° C. or more, 50° C. or more, 55° C. or more, 60° C. ormore, 65° C. or more, or 70° C. or more. The high temperature duringhydrolysis has many advantages, which include working at the optimumtemperature of the enzyme composition, the reduction of risk of(bacterial) contamination, reduced viscosity, smaller amount of coolingwater required, use of cooling water with a higher temperature, re-useof the enzymes and more.

In a further aspect of the invention, the amount of enzyme compositionadded (herein also called enzyme dosage or enzyme load) is low. In anembodiment the amount of enzyme is 6 mg protein/g dry matter weight orlower, 5 mg protein/g dry matter or lower, 4 mg protein/g dry matter orlower, 3 mg protein/g dry matter or lower, 2 mg protein/g dry matter orlower, or 1 mg protein/g dry matter or lower (expressed as protein in mgprotein/g dry matter). In an embodiment, the amount of enzyme is 0.5 mgenzyme/g dry matter weight or lower, 0.4 mg enzyme composition/g drymatter weight or lower, 0.3 mg enzyme/g dry matter weight or lower, 0.25mg enzyme/g dry matter weight or lower, 0.20 mg enzyme/g dry matterweight or lower, 0.18 mg enzyme/g dry matter weight or lower, 0.15 mgenzyme/g dry matter weight or lower or 0.10 mg enzyme/g dry matterweight or lower (expressed as total of cellulase enzymes in mg enzyme/gdry matter). Low enzyme dosage is possible, since because of theactivity and stability of the enzymes, it is possible to increase thehydrolysis reaction time.

In a further aspect of the invention, the hydrolysis reaction time is 5hours or more, 10 hours or more, 20 hours or more, 40 hours or more, 50hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90hours or more, 100 hours or more, 120 hours or more, 130 h or more. Inanother aspect, the hydrolysis reaction time is 5 to 150 hours, 40 to130 hours, 50 to 120 hours, 60 to 120 hours, 60 to 110 hours, 60 to 100hours, 70 to 100 hours, 70 to 90 hours or 70 to 80 hours. Due to thestability of the enzyme composition, longer hydrolysis reaction timesare possible with corresponding higher sugar yields.

The pH during hydrolysis may be chosen by the skilled person. In afurther aspect of the invention, the pH during the hydrolysis may be 3.0to 6.4. The stable enzymes of the invention may have a broad pH range ofup to 2 pH units, up to 3 pH units, up to 5 pH units. The optimum pH maylie within the limits of pH 2.0 to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to6.0, 3.5 to 5.0, 3.5 to 4.5, 4.0 to 4.5 or is about 4.2.

In a further aspect of the invention the hydrolysis step is conducteduntil 70% or more, 80% or more, 85% or more, 90% or more, 92% or more,95% or more of available sugar in lignocellulosic material is released.

Significantly, a process of the invention may be carried out using highlevels of dry matter (of the lignocellulosic material) in the hydrolysisreaction. Thus, the invention may be carried out with a dry mattercontent of about 5 wt % or higher, about 8 wt % or higher, about 10 wt %or higher, about 11 wt % or higher, about 12 wt % or higher, about 13 wt% or higher, about 14 wt % or higher, about 15 wt % or higher, about 20wt % or higher, about 25 wt % or higher, about 30 wt % or higher, about35 wt % or higher or about 40 wt % or higher. In a further embodiment,the dry matter content in the hydrolysis step is 14 wt %, 15 wt %, 16 wt%, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt%, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt%, 33 wt % or more or 14 to 33 wt %. In another embodiment the drymatter content at the end of the hydrolysis is 5 wt % or higher, 6 wt %or higher, 7 wt % or higher, 8 wt % or higher, 9 wt % or higher, 10 wt %or higher, 11 wt % or higher, 12 wt % or higher, 13 wt % or higher, 14wt % or higher, 15 wt % or higher, 16 wt % or higher, 17 wt % or higher,18 wt % or higher, 19 wt % or higher, 20 wt % or higher, 21 wt % orhigher, 22 wt % or higher, 23 wt % or higher, 24 wt % or higher, 25 wt %or higher, 26 wt % or higher, 27 wt % or higher, 28 wt % or higher, 29wt % or higher, 30 wt % or higher, 31 wt % or higher, 32 wt % or higher,33 wt % or higher, 34 wt % or higher, 35 wt % or higher, 36 wt % orhigher, 37 wt % or higher, 38 wt % or higher or 39 wt % or higher. Inanother embodiment the dry matter content at the end of the enzymatichydrolysis is between 5 wt %-40 wt %, 6 wt %-40 wt %, 7 wt %-40 wt %, 8wt %-40 wt %, 9 wt %-40 wt %, 10 wt %-40 wt %, 11 wt %-40 wt %, 12 wt%-40 wt %, 13 wt %-40 wt %, 14 wt %-40 wt %, 15 wt %-40 wt %, 16 wt %-40wt %, 17 wt %-40 wt %, 18 wt %-40 wt %, 19 wt %-40 wt %, 20 wt %-40 wt%, 21 wt %-40 wt %, 22 wt %-40 wt %, 23 wt %-40 wt %, 24 wt %-40 wt %,25 wt %-40 wt %, 26 wt %-40 wt %, 27 wt %-40 wt %, 28 wt %-40 wt %, 29wt %-40 wt %, 30 wt %-40 wt %, 31 wt %-40 wt %, 32 wt %-40 wt %, 33 wt%-40 wt %, 34 wt %-40 wt %, 35 wt %-40 wt %, 36 wt %-40 wt %, 37 wt %-40wt %, 38 wt %-40 wt %, 39 wt %-40 wt %.

Fermentation

The process according to the invention comprises a fermentation step. Ina further aspect, the invention thus includes in step fermentationprocesses in which a microorganism is used for the fermentation of acarbon source comprising sugar(s), e.g. glucose, L-arabinose and/orxylose. The carbon source may include any carbohydrate oligo- or polymercomprising L-arabinose, xylose or glucose units, such as e.g.lignocellulose, xylans, cellulose, starch, arabinan and the like. Forrelease of xylose or glucose units from such carbohydrates, appropriatecarbohydrases (such as xylanases, glucanases, amylases and the like) maybe added to the fermentation medium or may be produced by the modifiedhost cell. In the latter case, the modified host cell may be geneticallyengineered to produce and excrete such carbohydrases. An additionaladvantage of using oligo- or polymeric sources of glucose is that itenables to maintain a low(er) concentration of free glucose during thefermentation, e.g. by using rate-limiting amounts of the carbohydrases.This, in turn, will prevent repression of systems required formetabolism and transport of non-glucose sugars such as xylose. In apreferred process the modified host cell ferments both the L-arabinose(optionally xylose) and glucose, preferably simultaneously in which casepreferably a modified host cell is used which is insensitive to glucoserepression to prevent diauxic growth. In addition to a source ofL-arabinose, optionally xylose (and glucose) as carbon source, thefermentation medium will further comprise the appropriate ingredientrequired for growth of the modified host cell. Compositions offermentation media for growth of microorganisms such as yeasts orfilamentous fungi are well known in the art.

The fermentation time may be shorter than in conventional fermentationat the same conditions, wherein part of the enzymatic hydrolysis stillhas to take part during fermentation. In one embodiment, thefermentation time is 100 hours or less, 90 hours or less, 80 hours orless, 70 hours or less, or 60 hours or less, for a sugar composition of50 g/l glucose and corresponding other sugars from the lignocellulosicfeedstock (e.g. 50 g/l xylose, 35 g/l L-arabinose and 10 g/l galactose.For more dilute sugar compositions, the fermentation time maycorrespondingly be reduced.

The fermentation process may be an aerobic or an anaerobic fermentationprocess. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable), and wherein organic molecules serve asboth electron donor and electron acceptors. In the absence of oxygen,NADH produced in glycolysis and biomass formation, cannot be oxidised byoxidative phosphorylation. To solve this problem many microorganisms usepyruvate or one of its derivatives as an electron and hydrogen acceptorthereby regenerating NAD⁺. Thus, in a preferred anaerobic fermentationprocess pyruvate is used as an electron (and hydrogen acceptor) and isreduced to fermentation products such as ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotics and a cephalosporin.In a preferred embodiment, the fermentation process is anaerobic. Ananaerobic process is advantageous since it is cheaper than aerobicprocesses: less special equipment is needed. Furthermore, anaerobicprocesses are expected to give a higher product yield than aerobicprocesses. Under aerobic conditions, usually the biomass yield is higherthan under anaerobic conditions. As a consequence, usually under aerobicconditions, the expected product yield is lower than under anaerobicconditions.

In another embodiment, the fermentation process is under oxygen-limitedconditions. More preferably, the fermentation process is aerobic andunder oxygen-limited conditions. An oxygen-limited fermentation processis a process in which the oxygen consumption is limited by the oxygentransfer from the gas to the liquid. The degree of oxygen limitation isdetermined by the amount and composition of the ingoing gas flow as wellas the actual mixing/mass transfer properties of the fermentationequipment used. Preferably, in a process under oxygen-limitedconditions, the rate of oxygen consumption is at least 5.5, morepreferably at least 6 and even more preferably at least 7 mmol/L/h.

The fermentation process is preferably run at a temperature that isoptimal for the modified cell. Thus, for most yeasts or fungal cells,the fermentation process is performed at a temperature which is lessthan 42° C., preferably less than 38° C. For yeast or filamentous fungalhost cells, the fermentation process is preferably performed at atemperature which is lower than 35, 33, 30 or 28° C. and at atemperature which is higher than 20, 22, or 25° C.

In an embodiment of the invention, in step the fermentation is conductedwith a microorganism that is able to ferment at least one C5 sugar. Inan embodiment the process is a process for the production of ethanolwhereby the process comprises the step comprises fermenting a mediumcontaining sugar(s) with a microorganism that is able to ferment atleast one C5 sugar, whereby the host cell is able to ferment glucose,L-arabinose and xylose to ethanol. The microorganism may be aprokaryotic or eukaryotic organism. The microorganism used in theprocess may be a genetically engineered microorganism. Examples ofsuitable organisms are yeasts, for instance Saccharomyces, e.g.Saccharomyces cerevisiae, Saccharomyces pastorianus or Saccharomycesuvarum, Hansenula, Issatchenkia, e.g. Issatchenkia orientalis, Pichia,e.g. Pichia stipites or Pichia pastoris, Kluyveromyces, e.g.Kluyveromyces fagilis, Candida, e.g. Candida pseudotropicalis or Candidaacidothermophilum, Pachysolen, e.g. Pachysolen tannophilus or bacteria,for instance Lactobacillus, e.g. Lactobacillus lactis, Geobacillus,Zymomonas, e.g. Zymomonas mobilis, Clostridium, e.g. Clostridiumphytofermentans, Escherichia, e.g. E. coli, Klebsiella, e.g. Klebsiellaoxytoca. In an embodiment thereof the microorganism that is able toferment at least one C5 sugar is a yeast. In an embodiment, the yeast isbelongs to the genus Saccharomyces, preferably of the speciesSaccharomyces cerevisiae, in which genetic modifications have been made.An example of such a microorganism and its preparation is described inmore detail in WO 2008/041840 and in European Patent ApplicationEP10160622.6, filed 21 Apr. 2010. In an embodiment, the fermentationprocess for the production of ethanol is anaerobic. Anaerobic hasalready been defined earlier herein. In another preferred embodiment,the fermentation process for the production of ethanol is aerobic. Inanother preferred embodiment, the fermentation process for theproduction of ethanol is under oxygen-limited conditions, morepreferably aerobic and under oxygen-limited conditions. Oxygen-limitedconditions have already been defined earlier herein.

In such process, the volumetric ethanol productivity is preferably atleast 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre perhour. The ethanol yield on L-arabinose and optionally xylose and/orglucose in the process preferably is at least 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is herein defined as apercentage of the theoretical maximum yield, which, for glucose andL-arabinose and optionally xylose is 0.51 g. ethanol per g. glucose orxylose.

In one aspect, the fermentation process leading to the production ofethanol, has several advantages by comparison to known ethanolfermentations processes:

-   -   anaerobic processes are possible;    -   oxygen limited conditions are also possible;    -   higher ethanol yields and ethanol production rates can be        obtained;    -   the strain used may be able to use L-arabinose and optionally        xylose.

Alternatively to the fermentation processes described above, at leasttwo distinct cells may be used, this means this process is aco-fermentation process. All preferred embodiments of the fermentationprocesses as described above are also preferred embodiments of thisco-fermentation process: identity of the fermentation product, identityof source of L-arabinose and source of xylose, conditions offermentation (aerobic or anaerobic conditions, oxygen-limitedconditions, temperature at which the process is being carried out,productivity of ethanol, yield of ethanol).

The fermentation process may be carried out without any requirement toadjust the pH during the process. That is to say, the process is onewhich may be carried out without the addition of any acid(s) or base(s).However, this excludes a pretreatment step, where acid may be added. Thepoint is that the composition of the invention is capable of acting atlow pH and, therefore, there is no need to adjust the pH of acid of anacid pretreated feedstock in order that saccharification or hydrolysismay take place. Accordingly, a method of the invention may be a zerowaste method using only organic products with no requirement forinorganic chemical input.

Overall Reaction Time

According to the invention, the overall reaction time (or the reactiontime of hydrolysis step and fermentation step together) may be reduced.In one embodiment, the overall reaction time is 300 hours or less, 200hours or less, 150 hours or less, 140 hours or less, 130 or less, 120hours or less, 110 hours or less, 100 hours of less, 90 hours or less,80 hours or less, 75 hours or less, or about 72 hours at 90% glucoseyield. Correspondingly lower overall times may be reached at lowerglucose yield.

Fermentation Products

Fermentation products which may be produced according to the inventioninclude amino acids, vitamins, pharmaceuticals, animal feed supplements,specialty chemicals, chemical feedstocks, plastics, solvents, fuels, orother organic polymers, lactic acid, and ethanol, including fuel ethanol(the term “ethanol” being understood to include ethyl alcohol ormixtures of ethyl alcohol and water).

Specific value-added products that may be produced by the methods of theinvention include, but not limited to, biofuels (including biogas,ethanol and butanol); lactic acid; 3-hydroxy-propionic acid; acrylicacid; acetic acid; 1,3-propane-diol; ethylene; glycerol; a plastic; aspecialty chemical; an organic acid, including citric acid, succinicacid and maleic acid; a solvent; an animal feed supplement; apharmaceutical such as a β-lactam antibiotic or a cephalosporin; avitamin; an amino acid, such as lysine, methionine, tryptophan,threonine, and aspartic acid; an enzyme, such as a protease, acellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, anoxidoreductase, a transferase or a xylanase; a chemical feedstock; or ananimal feed supplement.

Fermentation products that may be produced by the processes of theinvention can be any substance derived from fermentation, They include,but are not limited to, alcohols (such as arabinitol, butanol, ethanol,glycerol, methanol, 1,3-propanediol, sorbitol, and xylitol); organicacid (such as acetic acid, acetonic acid, adipic acid, ascorbic acid,acrylic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, maleic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); ketones (such as acetone); amino acids(such as aspartic acid, glutamic acid, glycine, lysine, serine,tryptophan, and threonine); alkanes (such as pentane, hexane, heptane,octane, nonane, decane, undecane, and dodecane), cycloalkanes (such ascyclopentane, cyclohexane, cycloheptane, and cyclooctane), alkenes (suchas pentene, hexene, heptene, and octene); and gases (such as methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be a protein, a vitamin, a pharmaceutical,an animal feed supplement, a specialty chemical, a chemical feedstock, aplastic, a solvent, ethylene, an enzyme, such as a protease, acellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, anoxidoreductase, a transferase or a xylanase.

Separation of Fermentation Product

The process according to the invention optionally comprises recovery offermentation product. A fermentation product may be separated from thefermentation broth in any known manner. For each fermentation productthe skilled person will thus be able to select a proper separationtechnique. For instance ethanol may be separated from a yeastfermentation broth by distillation, for instance steamdistillation/vacuum distillation in conventional way.

Certain embodiments of the invention will below be described in moredetail, but are in no way limiting the scope of the present invention.

Use of Thermostable Enzymes Under Optimal Temperature Conditions

In one embodiment, the invention relates to the use of thermostableenzymes such as cellulolytic enzymes of Rasamsonia for the production ofreducing sugars from pre-treated lignocellulosic feedstock in, but notlimiting to, ethanol production. Cellulolytic enzymes of Rasamsoniaapplied on pre-treated lignocellulosic feedstock showed maximalconversion rates at temperature within the range of 50 to 70° C. Theenzyme remains active under these circumstances for 14 days and morewithout complete cessation of activity.

By using optimal temperature conditions, maximal amount of reducingsugars can be released from feedstock (total hydrolysis) within theshortest possible hydrolysis time. In this way, 100% conversion ofcellulose in glucose is achieved in less than 5 days.

The theoretical maximum yield (Yps max in g product per gram glucose) ofa fermentation product can be derived from textbook biochemistry. Forethanol, 1 mole of glucose (180 g) yields according to normal glycolysisfermentation pathway in yeast 2 moles of ethanol (=2×46=92 g ethanol.The theoretical maximum yield of ethanol on glucose is therefore92/180=0.511 g ethanol/g glucose.

For butanol (MW 74 g/mole) or iso-butanol, the theoretical maximum yieldis 1 mole of butanol per mole of glucose. So Yps max for(iso-)butanol=74/180=0.411 g (iso-) butanol/g glucose.

For lactic acid the fermentation yield for homolactic fermentation is 2moles of lactic acid (MW=90 g/mole) per mole of glucose. According tothis stoichiometry, the Yps max=1 g lactic acid/g glucose.

For other fermentation products a similar calculation may be made.

The cost reduction achieved with applying cellulolytic enzymes ofRasamsonia will be the result of an overall process time reduction.

Compensation of Lower Enzyme Dosage with Extended Hydrolysis Time UsingRasamsonia Enzymes

Due to the high stability of the stable enzymes, the activities do notcease in time, although less reducing sugars are liberated in the courseof the hydrolysis. It is possible to lower the enzyme dosage and extendthe use of the enzyme by prolonging the hydrolysis times to obtainsimilar levels of released reducing sugars. For example, 0.175 mLenzyme/g feedstock dry-matter resulted in release of approximately 90%of the theoretical maximum of reducing sugars from pre-treated feedstockwithin 72 h. When using 0.075 mL enzyme/g feedstock dry-matter,approximately 90% conversion of the theoretical maximum is achievedwithin 120 h. The results show that, because of the stability of theenzyme activity, lowering the enzyme dosage can be compensated byextending the hydrolysis time to obtain the same amount of reducingsugars. The same holds for hydrolysis of pre-treated feedstock atdry-matter contents higher than 10% shows that compensating effect ofextended hydrolysis time at 15% dry matter feedstock.

The cost reduction achieved by using stable cellulolytic enzymes, suchas of Rasamsonia, results from requiring less enzyme dosage, resultingin similar hydrolysis conversion yields.

Lowering the Risk on Contamination with Stable Enzymes

In a common process for converting lignocellulosic material intoethanol, process steps are preferably done under septic conditions tolower the operational costs. Contamination and growth of contaminatingmicroorganisms can therefore occur and result in undesirable sideeffects, such lactic acid, formic acid and acetic acid production, yieldlosses of ethanol on substrate, production of toxins and extracellularpolysaccharides, which may affect production costs significantly. A highprocess temperature and/or a short process time will limit the risk oncontamination during hydrolysis and fermentation. Thermostable enzymes,like those of Rasamsonia, are capable of hydrolysing lignocellulosicfeedstock at temperatures of higher than 60° C. At these temperatures,the risk that a contaminating microorganism will cause undesired sideeffects will be little to almost zero.

During the fermentation step, in which ethanol is produced, temperaturesare typically between 30 to 37° C. and will preferably not be raisedbecause of production losses. By applying fermentation process times asshort as possible the risks and effects of contamination and/or growthof contaminants will be reduced as much as possible. With stableenzymes, like those of Rasamsonia, a short as possible fermentationtimes can be applied (see description above), and thus risks oncontamination and/or growth of contaminants will be reduced as much aspossible. The cost reduction achieved with applying thermostablecellulolytic enzymes of Rasamsonia in this way will result from lowerrisk of process failures due to contamination.

Stable Enzymes Reduce Cooling Costs and Increase Productivity of EthanolPlants

The first step after thermal pretreatment will be to cool the pretreatedfeedstock to temperatures where the enzymes are optimal active. On largescale, this is typically done by adding (cooled) water, which will,besides decreasing the temperature, reduce the dry-matter content. Byusing thermos stable enzymes, like those of Rasamsonia, cost reductioncan be achieved by the fact that (i) less cooling of the pretreatedfeedstock is required since higher temperatures are allowed duringhydrolysis, and (ii) less water will be added, which will increase thedry-matter content during hydrolysis and fermentation and thus increasethe ethanol production capacity (amount produced per time unit pervolume) of an ethanol plant. Also, by using thermostable enzymesaccording to the invention, like those of Rasamsonia, cost reduction mayalso be achieved by using cooling water having higher temperature thatthe water that is used in a process with non-thermostable enzyme.

Enzyme Recycling after Hydrolysis with Stable Enzymes

At the end of the hydrolysis, enzyme activities appear to be low sincelittle reducing sugars are released once almost all cellulose isconverted. The amount of enzymatic activity present, however, hasdecreased only a little, assumingly mainly due to absorption of theenzymes to the substrate. By applying solid-liquid separation afterhydrolysis, such as centrifugation, filtration, sedicantation, etcetera,60% or more e.g. 70% of the enzyme activity in solution can be recoveredand re-used for hydrolysis of a new pre-treated lignocellulosicfeedstock during the next hydrolysis.

Moreover, after solid-liquid separation the enzyme in solution can beseparated from the solution containing reducing sugars and otherhydrolysis products from the enzymatic actions. This separation can bedone by, but not limiting to, (ultra and micro)filtration,centrifugation, sedicantation, sedimentation, with or without firstadsorption of the enzyme to a carrier of any kind.

For example, after hydrolysis of pre-treated feedstock with 0.175 mL/gfeedstock dry matter enzyme load for 20h, 50% of the theoretical maximumamount of reducing sugars is liberated and after the same hydrolysis for72h, 90% of the theoretical maximum amount of reducing sugars isliberated. By centrifugation and ultrafiltration, 60-70% of the enzymeactivity was recovered in the retentate, while the filtrate containedmore than 80% of the liberated reducing sugars. By re-using theretentate, either as it is or after further purification and/orconcentration, enzyme dosage during the next hydrolysis step can bereduced with 60 to 70%. The cost reduction achieved by using stablecellulolytic enzymes, such as of Rasamsonia, in this way results fromrequiring less enzyme dosage.

Enzyme Recycling after Hydrolysis in Combination with Enzyme Productionand Yeast-Cell Recycling with Stable Enzymes

The process including enzyme recycling after hydrolysis, as describedabove, can be combined with recycling of the ethanol producingmicroorganism after fermentation and with the use of the reducing sugarscontaining filtrate as a substrate (purified and/or concentrated ordiluted) in enzyme-production fermentation and as substrate for thecultivation of the ethanol-producing microorganism.

Enzyme Recycling after Vacuum Distillation with Stable Enzymes

The thermo stability of enzymes, like those from Rasamsonia-causesremaining cellulolytic activity after hydrolysis, fermentation andvacuum distillation in the thin stillage. The total activity of theenzyme is reduced during the three successive process steps. The thinstillage obtained after vacuum distillation can thus be re-used as asource of enzyme for a newly startedhydrolysis-fermentation-distillation process cycle of pre-treated wheatstraw conversion into ethanol. The thin stillage can be used either inconcentrated or (un)diluted form and/or purified and with or withoutadditional enzyme supplementation.

Enzyme Recycling in Combination with Enzyme Supplementation after VacuumDistillation with Thermostable Enzymes

In an optimal process, an amount of enzyme is supplemented into the thinstillage, before its re-use in a new process cycle, equal to the amountof activity lost during the three successive process steps of theprevious process cycle. In this way over-dosage of enzyme is avoided andthus most efficient use of enzyme is obtained.

Moreover, by providing high enzyme dosage in the first process cycle,and supplementing enzyme equal to the amount of activity lost during thethree successive process steps in the following process cycles, highestpossible hydrolysis rates can be obtained in each process cycleresulting in short hydrolysis times of less than 48 h in combinationwith most efficient use of enzymes.

Use of Stable Enzymes in Mixed Systems

By applying mixing during hydrolysis, enzymes come more often in contactwith substrates, which results in a more efficient use of the catalyticactivity. This will result in a lower enzyme dosages and thus in lowercosts, unless the mixing has a negative effect on the enzymes. Stableenzymes, like the thermostable enzymes from Rasamsonia, are robust andcan resist circumstances of (locally) high shear and temperatures, whichis the case during intensive mixing of slurries. The use of it in mixedsystems is therefore beneficial and will lead to dosage and thus costsreduction.

The invention is further described by the following examples, whichshould not be construed as limiting the scope of the invention.

EXAMPLES Experimental Information Strains

Rasamsonia (Talaromyces) emersonii strain was deposited at CENTRAALBUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 ADUtrecht, The Netherlands in December 1964 having the Accession NumberCBS 393.64. Other suitable strains can be equally used in the presentexamples to show the effect and advantages of the invention. For exampleTEC-101, TEC-147, TEC-192, TEC-201 or TEC-210 are suitable Rasamsoniastrains which are described in WO 2011/000949.

Preparation of Acid Pre-Treated Corn Stover Substrate.

Dilute-acid pre-treated corn stover (aCS) was obtained as described inSchell, D. J., Applied Biochemistry and Biotechnology (2003), vol.105-108, pp 69-85. A pilot scale pretreatment reactor was used operatingat steady state conditions of 190° C., 1 min residence time and aneffective H₂SO₄ acid concentration of 1.45% (w/w) in the liquid phase.

Protein Measurement Assays 1. Total Protein TCA Biuret

The method was a combination of precipitation of protein usingtrichloro-acetic acid (TCA) to remove disturbing substances and allowdetermination of the protein concentration with the colorimetric Biuretreaction. In the Biuret reaction, a copper (II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptidebonds in an alkaline solution. A violet color indicates the presence ofproteins. The intensity of the color, and hence the absorption at 546nm, is directly proportional to the protein concentration, according tothe Beer-Lambert law. The standardisation was performed using BSA(Bovine Serum Albumine) and the protein content was expressed in gprotein as BSA equivalent/L or mg protein as BSA equivalent/ml. Theprotein content was calculated using standard calculation protocolsknown in the art, by plotting the OD₅₄₆ versus the concentration ofsamples with known concentration, followed by the calculation of theconcentration of the unknown samples using the equation generated fromthe calibration line.

2. Individual Proteins Using PAGE Sample Pre-Treatment SDS-PAGE

Based on the estimated protein concentration of the samples thefollowing samples preparation was performed. To 10 μl sample 40 μlMilliQ water and 50 μl TCA (20%) was added to dilute the sample fivetimes (˜1 mg/ml) and precipitate the proteins. After 1 hour on ice thesample was centrifuged (10 minutes, 14000 rpm). The pellet was washedwith 500 μl aceton and centrifuged (10 minutes, 14000 rpm). The pelletwas treated as described below.

SDS-Page

The pellet was dissolved in 65 μl of the MilliQ water, 25 μl NuPAGE™ LDSsample buffer (4×) Invitrogen and 10 μl NuPAGE™ Sample Reducing agent(10×) Invitrogen. Prior to the denaturation step the sample was diluted5 times using a mix of MilliQ; NuPAGE™ LDS sample buffer and 10 μlNuPAGE™ Sample Reducing in the ratio of 65:25:10. After mixing, thesamples were incubated in a thermo mixer for 10 minutes at 70° C. Thesample solutions were applied on a 4-12% Bis-Tris gel (NuPAGE™ BisTris,Invitrogen). A sample (10 μl) of marker M12 (Invitrogen) was alsoapplied on the gel. The gel was run at 200 V for 50 minutes, using theXCELL Surelock, with 600 ml 20× diluted SDS buffer in the outer bufferchamber and 200 ml 20× diluted SDS buffer, containing 0.5 ml ofantioxidant (NuPAGE™ Invitrogen) in the inner buffer chamber. Afterrunning, the gel was rinsed twice with demineralised water the gels werefixed with 50% methanol/7% acetic acid solution for one hour and stainedwith Sypro Ruby (50 ml per gel) overnight. An image was made using theTyphoon 9200 (610 BP 30, Green (532 nm), PMT 600V, 100 micron) afterwashing the gel with MilliQ water.

Quantitative Analysis of the Protein

Using the Typhoon scanner the ratio between protein bands within a lanewas determined using standard methods known in the art. The sample wasapplied in triplicate and the grey values were determined using theprogram Image quant. Values are expressed as relative % protein to thetotal protein, calculated using the gray value of the selected proteinband relative to the total gray value all the protein bands.

Glucan Conversion Calculation:

% glucan conversion (%)=(glucose(g/l)×100%)/(glucan(fraction onDM)×dm(g/kg)×1.1)

Wherein:

glucose (g/l)=glucose concentration in supernatant after hydrolysis.glucan (fraction on dm)=glucan content of the substrate beforepretreatment.dm (g/kg)=dry matter of hydrolysis (f.i. 20% dm=200 g/kg).1.1=weight increase due to water incorporation during hydrolysis.

Example Calculation:

glucose=60 g/Iglucan fraction=0.40 (is 40% on dry matter)dm=200 g/kg

glucan conversion example=(60*100)/(0.4×200×1.1)=68% conversion

Example 1 Evaluation of the Effect of the Absence of Oxygen DuringHydrolysis on the Cellulolytic Activity of Cellulase Enzyme Cocktails

The effect of oxygen absence during hydrolysis on the cellulolyticactivity of the enzyme cocktail was evaluated according to theprocedures described below. The hydrolysis reactions were performed withacid pretreated cornstover (aCS) feedstock at a final concentration of10 w/w % DM. This feedstock solution was prepared via the dilution of aconcentrated feedstock solution with water. Subsequently the pH wasadjusted to pH 4.5 with a 4M NaOH solution. The elimination of oxygenfrom the feedstock was accomplished in two steps. First, the feedstocksolution was degassed via sonication under vacuum in a sonication bath(Bransonic 5510E-DTH, setting; Degas) for 15 minutes. In the secondstep, the oxygen was further removed by continuous sparging of anitrogen flow through a 500 ml solution of the 10% DM feedstock for aperiod of 3 hours. Prior to being sparged through the feedstocksolution, the nitrogen flow was sparged through water in order tosaturate it with water vapour and prevent evaporation of the water fromthe feedstock solution. In parallel, 500 ml of the same batch 10 w/w %DM aCS was sparged with air as an oxygen-containing control sample in asimilar set-up and according to the same protocol.

The hydrolysis of the oxygen-depleted (nitrogen sparged) and theoxygen-saturated (air-sparged) 10 w/w % aCS feedstock solutions wereconducted in air-tight, 30-ml centrifuge bottles (Nalgene Oakridge) in atotal reaction volume of 10 ml. The bottles, already containing thecellulase solution, used for the oxygen-depleted experiment were spargedwith nitrogen prior to- and during filling them with feedstock. Eachhydrolysis was performed in duplicate with 7.5 mg/g DM cellulase enzymecocktail added in a total volume not larger than 375 μl. TEC-210 wasfermented according to the inoculation and fermentation proceduresdescribed in WO2011/000949.

The centrifuge bottles containing the feedstock and enzyme solution wereplaced in an oven incubator (Techne HB-1 D hybridization oven) andincubated for 72 hours at 65° C. while rotating at set-point 3 (12 rpmper minute). Following hydrolysis, the samples were cooled on ice andimmediately 50 μl of each supernatant was diluted in 1450 μl grade Iwater. The diluted supernatant was subsequently filtered (0.45 μmfilter, Pall PN 454) and the filtrates were analysed for sugar contentas described below.

The sugar concentrations of the diluted samples were measured using anHPLC equipped with an Aminex HPX-87P column (Biorad #1250098) by elutionwith water at 85° C. at a flow rate of 0.6 ml per minute and quantifiedby integration of the glucose signals from refractive index detection(R.I.) calibrated with glucose standard solutions.

The data presented in Table 1/FIG. 1 show that the glucose released fromthe nitrogen-sparged feedstocks is lower than the glucose released fromthe feedstocks sparged with air.

Based on these results we conclude that the presence of oxygen improvesthe cellulolytic performance of cellulase mixtures.

TABLE 1 The effect of sparging nitrogen or air through a 10% aCSfeedstock before hydrolysis, on the total amount of glucose released.Cellulase Sparged with air Sparged with N₂ cocktail Average glucose(g/l) stdev Average glucose (g/l) stdev TEC-210 34.5 0.8 31.9 1.1

Example 2 The Effect of Oxygen on the Cellulolytic Activity of CellulaseEnzyme Cocktails During Hydrolysis of Lignocellulosic Feedstock

The effect of oxygen on the cellulolytic activity of the enzyme cocktailduring the hydrolysis of lignocellulosic feedstock is shown in thisexample. The hydrolysis reactions are performed with acid pretreatedcornstover (aCS) feedstock at a final concentration of 20 w/w % DM. Thisfeedstock solution is prepared via the dilution of a concentratedfeedstock solution with water. Subsequently the pH is adjusted to pH 4.5with a 10% (w/w) NH₄OH solution.

The hydrolysis is done in a stirred, pH controlled and temperaturecontrolled reactor with a working volume of 1 l. Each hydrolysis isperformed in duplicate with 2.5 mg/g DM TEC-210 cellulase enzymecocktail. TEC-210 was produced according to the inoculation andfermentation procedures described in WO2011/000949.

The following experiments are done:

-   1. 1 l of 20% aCS, pH 4.5, temperature 62° C., stirrer speed 60 rpm    (this corresponds with a DO level of <0.002 mol of oxygen per m³),    2.5 mg/g dm TEC-210 cellulase cocktail, incubation time 120 hours    (reference experiment).-   2. As experiment 1 but at the start of the hydrolysis, air sparging    into the solution started to a dissolved oxygen level of 20% (this    corresponds to 0.03 mol of oxygen per m3, measured using a DO    (dissolved oxygen) electrode). This dissolved oxygen level is    maintained throughout the rest of the hydrolysis process.-   3. As experiment 1 but at 72 hours air sparging into the solution    started to a dissolved oxygen level of 20% (this corresponds to 0.03    mol of oxygen per m3, measured using a DO (dissolved oxygen)    electrode). This dissolved oxygen level is maintained throughout the    rest of the hydrolysis process.

After the hydrolysis, the samples are cooled on ice and immediately 50μl of each supernatant is diluted in 1450 μl grade I water. The dilutedsupernatant is subsequently filtered (0.45 μm filter, Pall PN 454) andthe filtrates are analysed for sugar content as described below.

The sugar concentrations of the diluted samples are measured using anHPLC equipped with an Aminex HPX-87P column (Biorad #1250098) by elutionwith water at 85° C. at a flow rate of 0.6 ml per minute and quantifiedby integration of the glucose signals from refractive index detection(R.I.) calibrated with glucose standard solutions. Results, visible inFIG. 2 clearly show an increased glucose production in case air isadded. In addition, air added to the hydrolysis reaction in the secondpart of the time demonstrates superior glucose production compared to noair addition or an air addition during the whole hydrolysis step.

Example 3 The Effect of Partial Aeration (in Time) on the EnzymaticHydrolysis of Lignocellulosic Feedstock on Pilot Scale

The effect of the dissolved oxygen concentration on the cellulolyticactivity of the enzyme cocktail or composition during the hydrolysis oflignocellulosic feedstock on pilot scale is shown in this example. Thehydrolysis reactions are performed with acid pretreated cornstover (aCS)feedstock at a final concentration of 17.1 w/w % DM. The feedstocksolution is prepared by the dilution of concentrated feedstock slurrywith water. The pH is adjusted to pH 4.5 with a 25% (w/w) NH₄OHsolution.

The enzymatic hydrolysis is done in a 270 litre pilot reactor which ispH and temperature controlled with a working volume of 150 litre. Thedissolved oxygen during the process is controlled by adjusting impellerspeed at a given airflow and overpressure. The enzymatic hydrolysis isperformed at a dosage of 2.5 mg (TCA protein)/g dm TEC-210 cellulaseenzyme cocktail. TEC-210 was produced according to the inoculation andfermentation procedures described in WO2011/000949.

The following experiments are done:

Experiment 1

-   -   Aeration from 0 to 120 hours: 150 l of 17.1% pCS, pH 4.5,        temperature 62° C., 1 bar overpressure, 10 kg/h airflow in the        headspace, 2.5 mg TCA/g dm TEC-210 cellulase cocktail,        incubation time 120 hours in a 270 litre pilot reactor The        dissolved oxygen concentration (DO) of the reaction mixture was        measured constantly using a DO electrode. The DO was controlled        at a level of 0.15-0.22 mol/m³ by adjusting the impeller speed.

Experiment 2

-   -   Aeration between 72 and 120 hours: 150 l of 17.1% pCS, pH 4.5,        temperature 62° C., an enzyme dosage 2.5 mg TCA/g dm TEC-210        cellulase cocktail and a total incubation time of 120 hours in a        270 litre pilot reactor. The dissolved oxygen concentration (DO)        of the reaction mixture was measured constantly using a DO        electrode. For the first 72 hours of the process the following        settings were applied: no overpressure, no airflow in the        headspace and the DO was controlled at a level of [0.02-0.05]        mol/m3 by adjusting the impeller speed. For the last 48 hours of        the process the following settings were applied: 1 bar        overpressure, 10 kg/h airflow in the headspace and the DO was        controlled at a level of 0.15-0.22 mol/m³ by adjusting the        impeller speed.

During the enzymatic hydrolysis, samples were taken daily forcarbohydrate analysis (glucose, cellobiose) by NMR and viscosity and pHmeasurement.

Composition analysis of the pretreated Corn Stover was done by chemicalhydrolysis of the sample and determination of the mono saccharides byNMR.

Samples taken during enzymatic hydrolysis were analysed for(oligo)sugars, organic acids and inhibitors by flow NMR.

The results are presented in FIG. 4 and show that during enzymatichydrolysis in experiment 2 with the partial aeration (□=aeration betweenhydrolysis time is 72 and 120 hours) more glucose is produced thanduring enzymatic hydrolysis in experiment 1 (▪=aeration betweenhydrolysis time is 0 and 120 hours).

Example 4 The Effect of Timing of Dissolved Oxygen Supply on EnzymaticHydrolysis of Lignocellulosic Feedstock

The effect of timing of dissolved oxygen supply on the enzymatichydrolysis of lignocellulosic feedstock is shown in this example. Thehydrolysis reactions are performed with acid pretreated cornstover (aCS)feedstock at a final concentration of 20 w/w % DM. The feedstocksolution is prepared by the dilution of concentrated feedstock slurrywith water. The pH is adjusted to pH 4.5 with a 25% (w/w) NH₄OHsolution.

The enzymatic hydrolysis is done in a 2 litre reactor which is pH andtemperature controlled with a working volume of 1 litre. The dissolvedoxygen during the process is controlled by adjusting impeller speed andcontinuous refreshment of the headspace with fresh air in case of anincreased dissolved oxygen concentration. The enzymatic hydrolysis isperformed at a dosage of 1.5 mg (TCA protein)/g dm TEC-210 cellulaseenzyme cocktail. TEC-210 was produced according to the inoculation andfermentation procedures described in WO2011/000949.

The following experiments are done:

Experiment 1

Aeration from 0 to 7 hours: 1 l of 20% pCS, pH 4.5, temperature 62° C.,1.5 mg TCA/g dm TEC-210 cellulase cocktail, incubation time 120 hours.The dissolved oxygen concentration (DO) of the reaction mixture wasmeasured constantly using a DO electrode. The DO was controlled at alevel of >0.05 mol/m³ during the first 7 hours of the hydrolysisprocess. Between 7 and 120 hours of hydrolysis time the DO wasmaintained at a level <0.02 mol/m³.

Experiment 2

Aeration between 72 and 120 hours: 1 l of 20% pCS, pH 4.5, temperature62° C., 1.5 mg TCA/g dm TEC-210 cellulase cocktail, incubation time 120hours. The dissolved oxygen concentration (DO) of the reaction mixturewas measured constantly using a DO electrode. The DO was controlled at alevel of <0.01 mol/m³ during the first 72 hours of the hydrolysisprocess. Between 72 and 120 hours of hydrolysis time the DO wasmaintained at a level >0.05 mol/m³.

During the enzymatic hydrolysis, samples were taken daily forcarbohydrate analysis (glucose, cellobiose) by NMR and viscosity and pHmeasurement.

Composition analysis of the pretreated Corn Stover was done by chemicalhydrolysis of the sample and determination of the mono saccharides byNMR.

The results are presented in FIG. 5 and clearly demonstrate an increasein the glucose formation rate when the reaction mixture is aerated.Experiment 1, which was aerated between 0 and 7 hours, clearly shows anincreased glucose formation rate during the first 7 hours of the processcompared with the non-aerated situation during that process phase ofExperiment 2. In addition, Experiment 2 demonstrates an increasedglucose formation rate between 72 and 120 hours compared with thenon-aerated situation during that period in Experiment 1.

Example 5 The Effect of Timing of Dissolved Oxygen Supply on EnzymaticHydrolysis of Lignocellulosic Feedstock

Dilute-acid pre-treated corn stover (aCS) of 20% dry matter ishydrolysed in a reactor of 20 m³ working volume and a head space of 2 m³(reactor diameter: 2.5 m and reactor height: 4.5 m). Mixing in thereactor is done by gas recycle from the head space to a sparger at thebottom of the reactor with a gas flow of 100 m³/h. The gas flow isrealized with a compressor using a power input of 50 Watt/m³. Hydrolysisis performed with 2.5 mg/g DM TEC-210 cellulase enzyme cocktail. During120 hours in the reactor cellulose hydrolysis takes place at 62° C. andabout 1 g/I gluconic acid is formed. 1 g/I gluconic acid in the presentreactor corresponds with about 20 kg of gluconic acid or 102 Mol or 0.85moles per hour in case of a process time of 120 hours. This oxygendemand can be fulfilled by air supply at a rate of 90 l/h.

The recycle air flow of 100 m³/h is much higher than the supplied amountof fresh air of 90 l/h, so the freshly introduced air is diluted about1000 times. The diluted (fresh) air will be recycled through thehydrolysate, allowing for all oxygen to be transferred and consumed.

The exhaust gas flow corresponds to the inlet gas flow (minus oxygenconsumed). The oxygen in the gas flow will not exceed 0.01 mol/m³, whichwill keep the oxygen concentration in the hydrolysate low, and at thesame time exactly the desired amount of air is transported. In this wayvery small amounts of oxygen can be added to the system and the oxygenlevel can be controlled very accurately, even at very low oxygenconcentrations.

Example 6 The Influence of the Amount of Air on the Hydrolysis of Glucanin Lignocellulosic Feedstock

The enzymatic hydrolysis was performed using acid pretreated cornstover(aCS) feedstock at a concentration of 20% (w/w) dry matter (DM). Thefeedstock solution was prepared by the dilution of concentratedfeedstock slurry with water. The pH was adjusted to pH 4.5 with a 25%(w/w) NH₄OH-solution. The enzymatic hydrolysis was performed at 1 kgscale using a 1.5 liter reactor. The pH was controlled at 4.5 and thetemperature was controlled at 62° C. The dissolved oxygen during theprocess was controlled by headspace gas recycling and additional freshair (containing 20-21% oxygen).

Prior to enzyme addition, headspace gas was recylced at a gas flow of 3l/hour using a peristaltic pump and a sparger. Due to the fact that thefeedstock consumes oxygen through a chemical reaction, the DO levelreached a level of 0% DO within one hour resulting in an anaerobicfeedstock and a headspace which was completely depleted from oxygen. Theresulting inert headspace gas (oxygen free) was used throughout theentire hydrolysis as carrier gas for the introduced fresh air.

Next, the cellulase enzyme cocktail TEC210 was added to the feedstock ata dosage of 3.75 mg (TCA protein)/g dm. TEC-210 was produced accordingto the inoculation and fermentation procedures described inWO2011/000949. The total hydrolysis time was 120 hours.

Fresh air was introduced into the recycle loop of inert headspace gasduring the entire hydrolysis process at a fresh air flow of 0-3-6-12-24or 48 ml per kg reaction mixture per hour, respectively, startingdirectly after enzyme addition. The DO was measured constantly in allexperiments. Samples were drawn at the start and end of the experimentfor glucose analysis by HPLC.

A parallel experiment was conducted in a shake flask to determine themaximum level of glucan hydrolysis. This maximum hydrolysis wasdetermined by incubating the feedstock at a very high enzyme dosage (50mg (TCA protein)/g dm) at similar conditions (pH, temperature and dm) ina surplus of oxygen containing headspace air. DO measurement in eachexperiment constantly showed 0.0% DO. This can be explained by directconsumption of oxygen after its transfer from the oxygen containingrecycle gas stream into the liquid phase in the reactor.

The results are presented in Table 2 and clearly demonstrate acorrelation between the increase in glucose formation and the increasedintroduction of oxygen containing fresh air.

TABLE 2 The effect of addition of fresh air (oxygen) on the enzymatichydrolysis of lignocellulosic feedstock, visualized as production ofglucose. Fresh air flow (ml/kg/hour) 0 3 6 12 24 48 Glucose (in 14 14 1212 15 14 g/l) at start (t = 0 h) Glucose (in 46 50 55 54 56 59 g/l) atend (t = 120 h)

1. An apparatus which comprises: a) a reactor of at least 1 m³ whichcomprises a gas introducing means for introduction of gas in thereactor; b) optionally, at least one stirring means for stirring thereactor contents; c) a gas pump connected to the gas introducing meansfor introducing gas into the reactor; d) a recycle pipe connected at anend of the recycle pipe to the headspace of the reactor and connected atanother end of the recycle pipe to the gas pump; e) an exhaust connectedto the recycle pipe, for deleting gas from the reactor headspace; f) agas inlet for introducing fresh gas in the reactor, said gas inletconnected to the gas pump via the recycle pipe; g) a means forcontrolling the ratio between recycled gas and fresh gas.
 2. Theapparatus of claim 1, wherein the gas introducing means is a gas spargerthat is located adjacent to a bottom surface inside of the reactor. 3.The apparatus of claim 2, wherein the gas sparger is located adjacent toa bottom surface inside of the reactor.
 4. The apparatus of claim 3,wherein the gas sparger comprises nozzles or orifices for introductionof the gas into the reactor.
 5. The apparatus of claim 4, wherein thegas sparger nozzles or orifices comprise holes with a diameter between0.5 mm and 500 mm.
 6. The apparatus of claim 5, wherein the gas spargercovers a large part of the bottom surface inside of the reactor.
 7. Theapparatus of claim 1, wherein the means for controlling the ratiobetween gas recycled from the reactor headspace and fresh gas permitscontinuous addition of gas and discontinuous addition of gas.
 8. Theapparatus of claim 1, wherein the means for controlling the ratiobetween gas recycled from the reactor headspace and fresh gas is avalve.
 9. The apparatus of claim 1, comprising at least one stirringmeans for stirring the reactor contents.
 10. The apparatus of claim 1which does not comprise any stirring means for stirring the reactorcontents.
 11. The apparatus of claim 1, further comprising a venturitube for circulating a liquid portion of the reactor contents, wherebythe circulation of fluid through the venturi tube drives theintroduction of the gas into the reactor.